Antisense oligonucleotides for inducing exon skipping and methods of use thereof

ABSTRACT

An antisense molecule capable of binding to a selected target site to induce exon skipping in the dystrophin gene, as set forth in SEQ ID NO: 1 to 202.

FIELD OF THE INVENTION

The present invention relates to novel antisense compounds and compositions suitable for facilitating exon skipping. It also provides methods for inducing exon skipping using the novel antisense compounds as well as therapeutic compositions adapted for use in the methods of the invention.

BACKGROUND ART

Significant effort is currently being expended researching methods for suppressing or compensating for disease-causing mutations in genes. Antisense technologies are being developed using a range of chemistries to affect gene expression at a variety of different levels (transcription, splicing, stability, translation). Much of that research has focused on the use of antisense compounds to correct or compensate for abnormal or disease-associated genes in a myriad of different conditions.

Antisense molecules are able to inhibit gene expression with exquisite specificity and because of this many research efforts concerning oligonucleotides as modulators of gene expression have focused on inhibiting the expression of targeted genes such as oncogenes or viral genes. The antisense oligonucleotides are directed either against RNA (sense strand) or against DNA where they form triplex structures inhibiting transcription by RNA polymerase II. To achieve a desired effect in specific gene down-regulation, the oligonucleotides must either promote the decay of the targeted mRNA or block translation of that mRNA, thereby effectively preventing de novo synthesis of the undesirable target protein.

Such techniques are not useful where the object is to up-regulate production of the native protein or compensate for mutations which induce premature termination of translation such as nonsense or frame-shifting mutations. Furthermore, in cases where a normally functional protein is prematurely terminated because of mutations therein, a means for restoring some functional protein production through antisense technology has been shown to be possible through intervention during the splicing processes (Sierakowska H, et al., (1996) Proc Natl Acad Sci USA 93,12840-12844; Wilton S D, et al., (1999) Neuromusc Disorders 9, 330-338; van Deutekom J C et al., (2001) Human Mol Genet. 10, 1547-1554). In these cases, the defective gene transcript should not be subjected to targeted degradation so the antisense oligonucleotide chemistry should not promote target mRNA decay.

In a variety of genetic diseases, the effects of mutations on the eventual expression of a gene can be modulated through a process of targeted exon skipping during the splicing process. The splicing process is directed by complex multi-particle machinery that brings adjacent exon-intron junctions in pre-mRNA into close proximity and performs cleavage of phosphodiester bonds at the ends of the introns with their subsequent reformation between exons that are to be spliced together. This complex and highly precise process is mediated by sequence motifs in the pre-mRNA that are relatively short semi-conserved RNA segments to which bind the various nuclear splicing factors that are then involved in the splicing reactions. By changing the way the splicing machinery reads or recognises the motifs involved in pre-mRNA processing, it is possible to create differentially spliced mRNA molecules. It has now been recognised that the majority of human genes are alternatively spliced during normal gene expression, although the mechanisms invoked have not been identified. Using antisense oligonucleotides, it has been shown that errors and deficiencies in a coded mRNA could be bypassed or removed from the mature gene transcripts.

In nature, the extent of genetic deletion or exon skipping in the splicing process is not fully understood, although many instances have been documented to occur, generally at very low levels (Sherrat T G, et al., (1993) Am J Hum Genet. 53, 1007-1015). However, it is recognised that if exons associated with disease-causing mutations can be specifically deleted from some genes, a shortened protein product can sometimes be produced that has similar biological properties of the native protein or has sufficient biological activity to ameliorate the disease caused by mutations associated with the target exon (Lu Q L, et al., (2003) Nature Medicine 9, 1009-1014; Aartsma-Rus A et al., (2004) Am J Hum Genet. 74: 83-92).

This process of targeted exon skipping is likely to be particularly useful in long genes where there are many exons and introns, where there is redundancy in the genetic constitution of the exons or where a protein is able to function without one or more particular exons (e.g. with the dystrophin gene, which consists of 79 exons; or possibly some collagen genes which encode for repeated blocks of sequence or the huge nebulin or titin genes which are comprised of ˜80 and over 370 exons, respectively).

Efforts to redirect gene processing for the treatment of genetic diseases associated with truncations caused by mutations in various genes have focused on the use of antisense oligonucleotides that either: (1) fully or partially overlap with the elements involved in the splicing process; or (2) bind to the pre-mRNA at a position sufficiently close to the element to disrupt the binding and function of the splicing factors that would normally mediate a particular splicing reaction which occurs at that element (e.g., binds to the pre-mRNA at a position within 3, 6, or 9 nucleotides of the element to be blocked).

For example, modulation of mutant dystrophin pre-mRNA splicing with antisense oligoribonucleotides has been reported both in vitro and in vivo. In one type of dystrophin mutation reported in Japan, a 52-base pair deletion mutation causes exon 19 to be removed with the flanking introns during the splicing process (Matsuo et al., (1991) J Clin Invest. 87:2127-2131). An in vitro minigene splicing system has been used to show that a 31-mer 2′-O-methyl oligoribonucleotide complementary to the 5′ half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA (Takeshima et al. (1995), J. Clin. Invest., 95, 515-520). The same oligonucleotide was used to induce exon skipping from the native dystrophin gene transcript in human cultured lymphoblastoid cells.

Dunckley et al., (1997) Nucleosides & Nucleotides, 16, 1665-1668 described in vitro constructs for analysis of splicing around exon 23 of mutated dystrophin in the mdx mouse mutant, a model for muscular dystrophy. Plans to analyse these constructs in vitro using 2′ modified oligonucleotides targeted to splice sites within and adjacent to mouse dystrophin exon 23 were discussed, though no target sites or sequences were given.

2′-O-methyl oligoribonucleotides were subsequently reported to correct dystrophin deficiency in myoblasts from the mdx mouse from this group. An antisense oligonucleotide targeted to the 3′ splice site of murine dystrophin intron 22 was reported to cause skipping of the mutant exon as well as several flanking exons and created a novel in-frame dystrophin transcript with a novel internal deletion. This mutated dystrophin was expressed in 1-2% of antisense treated mdx myotubes. Use of other oligonucleotide modifications such as 2′-O-methoxyethyl phosphodiesters are described (Dunckley et al. (1998) Human Mol. Genetics, 5, 1083-90).

Thus, antisense molecules may provide a tool in the treatment of genetic disorders such as Duchenne Muscular Dystrophy (DMD). However, attempts to induce exon skipping using antisense molecules have had mixed success. Studies on dystrophin exon 19, where successful skipping of that exon from the dystrophin pre-mRNA was achieved using a variety of antisense molecules directed at the flanking splice sites or motifs within the exon involved in exon definition as described by Errington et al. (2003) J Gen Med 5, 518-527″.

In contrast to the apparent ease of exon 19 skipping, the first report of exon 23 skipping in the mdx mouse by Dunckley et al., (1998) is now considered to be reporting only a naturally occurring revertant transcript or artefact rather than any true antisense activity. In addition to not consistently generating transcripts missing exon 23, Dunckley et al., (1998) did not show any time course of induced exon skipping, or even titration of antisense oligonucleotides, to demonstrate dose dependent effects where the levels of exon skipping corresponded with increasing or decreasing amounts of antisense oligonucleotide. Furthermore, this work could not be replicated by other researchers.

The first example of specific and reproducible exon skipping in the mdx mouse model was reported by Wilton et al., (1999) Neuromuscular Disorders 9, 330-338. By directing an antisense molecule to the donor splice site, consistent and efficient exon 23 skipping was induced in the dystrophin mRNA within 6 hours of treatment of the cultured cells. Wilton et al., (1999), also describe targeting the acceptor region of the mouse dystrophin pre-mRNA with longer antisense oligonucleotides and being unable to repeat the published results of Dunckley et al., (1998). No exon skipping, either 23 alone or multiple removal of several flanking exons, could be reproducibly detected using a selection of antisense oligonucleotides directed at the acceptor splice site of intron 22.

While the first antisense oligonucleotide directed at the intron 23 donor splice site induced consistent exon skipping in primary cultured myoblasts, this compound was found to be much less efficient in immortalized cell cultures expressing higher levels of dystrophin. However, with refined targeting and antisense oligonucleotide design, the efficiency of specific exon removal was increased by almost an order of magnitude (see Mann C J et al., (2002) J Gen Med 4, 644-654).

Thus, there remains a need to provide antisense oligonucleotides capable of binding to and modifying the splicing of a target nucleotide sequence. Simply directing the antisense oligonucleotides to motifs presumed to be crucial for splicing is no guarantee of the efficacy of that compound in a therapeutic setting.

SUMMARY OF THE INVENTION

The present invention provides antisense molecule compounds and compositions suitable for binding to RNA motifs involved in the splicing of pre-mRNA that are able to induce specific and efficient exon skipping and a method for their use thereof.

The choice of target selection plays a crucial role in the efficiency of exon skipping and hence its subsequent application of a potential therapy. Simply designing antisense molecules to target regions of pre-mRNA presumed to be involved in splicing is no guarantee of inducing efficient and specific exon skipping. The most obvious or readily defined targets for splicing intervention are the donor and acceptor splice sites although there are less defined or conserved motifs including exonic splicing enhancers, silencing elements and branch points.

The acceptor and donor splice sites have consensus sequences of about 16 and 8 bases respectively (see FIG. 1 for schematic representation of motifs and domains involved in exon recognition, intron removal and the splicing process).

According to a first aspect, the invention provides antisense molecules capable of binding to a selected target to induce exon skipping.

For example, to induce exon skipping in exons 3 to 8, 10 to 16, 19 to 40, 42 to 44, 46, 47, and 50 to 53 in the Dystrophin gene transcript the antisense molecules are preferably selected from the group listed in Table 1A.

In a further example, it is possible to combine two or more antisense oligonucleotides of the present invention together to induce multiple exon skipping in exons 19-20, and 53. This is a similar concept to targeting of a single exon. A combination or “cocktail” of antisense oligonucleotides are directed at adjacent exons to induce efficient exon skipping.

In another example, to induce exon skipping in exons 19-20, 31, 34 and 53 it is possible to improve exon skipping of a single exon by joining together two or more antisense oligonucleotide molecules. This concept is termed by the inventor as a “weasel”, an example of a cunningly designed antisense oligonucleotide. A similar concept has been described in Aartsma-Rus A et al., (2004) Am J Hum Genet. 74: 83-92).

According to a second aspect, the present invention provides antisense molecules selected and or adapted to aid in the prophylactic or therapeutic treatment of a genetic disorder comprising at least an antisense molecule in a form suitable for delivery to a patient.

According to a third aspect, the invention provides a method for treating a patient suffering from a genetic disease wherein there is a mutation in a gene encoding a particular protein and the affect of the mutation can be abrogated by exon skipping, comprising the steps of: (a) selecting an antisense molecule in accordance with the methods described herein; and (b) administering the molecule to a patient in need of such treatment.

The invention also addresses the use of purified and isolated antisense oligonucleotides of the invention, for the manufacture of a medicament for treatment of a genetic disease.

The invention further provides a method of treating a condition characterised by Duchenne muscular dystrophy, which method comprises administering to a patient in need of treatment an effective amount of an appropriately designed antisense oligonucleotide of the invention, relevant to the particular genetic lesion in that patient. Further, the invention provides a method for prophylactically treating a patient to prevent or at least minimise Duchene muscular dystrophy, comprising the step of: administering to the patient an effective amount of an antisense oligonucleotide or a pharmaceutical composition comprising one or more of these biological molecules.

The invention also provides kits for treating a genetic disease, which kits comprise at least a antisense oligonucleotide of the present invention, packaged in a suitable container and instructions for its use.

Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description, which proceeds with reference to the following figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of motifs and domains involved in exon recognition, intron removal and the splicing process.

FIG. 2. Diagrammatic representation of the concept of antisense oligonucleotide induced exon skipping to by-pass disease-causing mutations (not drawn to scale). The hatched box represents an exon carrying a mutation that prevents the translation of the rest of the mRNA into a protein. The solid black bar represents an antisense oligonucleotide that prevents inclusion of that exon in the mature mRNA.

FIG. 3 Gel electrophoresis showing differing efficiencies of two antisense molecules directed at exon 8 acceptor splice site. The preferred compound [H8A(−06+18)] induces strong and consistent exon skipping at a transfection concentration of 20 nanomolar in cultured normal human muscle cells. The less preferred antisense oligonucleotide [H8A(−06+14)] also induces efficient exon skipping, but at much higher concentrations. Other antisense oligonucleotides directed at exon 8 either only induced lower levels of exon skipping or no detectable skipping at all (not shown).

FIG. 4 Gel electrophoresis showing differing efficiencies of two antisense molecules directed at internal domains within exon 7, presumably exon splicing enhancers. The preferred compound [H7A(+45+67)] induces strong and consistent exon skipping at a transfection concentration of 20 nanomolar in cultured human muscle cells. The less preferred antisense oligonucleotide [H7A(+2+26)] induces only low levels of exon skipping at the higher transfection concentrations. Other antisense oligonucleotides directed at exon 7 either only induced lower levels of exon skipping or no detectable skipping at all (not shown).

FIG. 5 Gel electrophoresis showing an example of low efficiency exon 6 skipping using two non-preferred antisense molecules directed at human exon 6 donor splice site. Levels of induced exon 6 skipping are either very low [H6D(+04−21)] or almost undetectable [H6D(+18−04)]. These are examples of non-preferred antisense oligonucleotides to demonstrate that antisense oligonucleotide design plays a crucial role in the efficacy of these compounds.

FIG. 6 Gel electrophoresis showing strong and efficient human exon 6 skipping using an antisense molecules [H6A(+69+91)] directed at an exon 6 internal domain, presumably an exon splicing enhancer. This preferred compound induces consistent exon skipping at a transfection concentration of 20 nanomolar in cultured human muscle cells.

FIG. 7 Gel electrophoresis showing strong human exon 4 skipping using an antisense molecule H4A(+13+32) directed at an exon 6 internal domain, presumably an exon splicing enhancer. This preferred compound induces strong and consistent exon skipping at a transfection concentration of 20 nanomolar in cultured human muscle cells.

FIG. 8 Gel electrophoresis showing (8B) strong human exon 11 skipping using antisense molecule H11A(+75+97) directed at an exon 11 internal domain; and (8B) strong human exon 12 skipping using antisense molecule H12A(+52+75) directed at exon 12 internal domain.

FIG. 9 Gel electrophoresis showing (9A) strong human exon 15 skipping using antisense molecules H15A(+48+71) and H15A(−12+19) directed at an exon 15 internal domain; and (9B) strong human exon 16 skipping using antisense molecules H16A(−12+19) and H16A(−06+25).

FIG. 10 Gel electrophoresis showing human exon 19/20 skipping using antisense molecules H20A(+44+71) and H20A(+149+170) directed at an exon 20 and a “cocktail” of antisense oligonucleotides H19A(+35+65, H20A(+44+71) and H20A(+149+170) directed at exons 19/20.

FIG. 11 Gel electrophoresis showing human exon 19/20 skipping using “weasels” directed at exons 19 and 20.

FIG. 12 Gel electrophoresis showing exon 22 skipping using antisense molecules H22A(+125+106), H22A(+47+69), H22A(+80+101) and H22D(+13−11) directed at exon 22.

FIG. 13 Gel electrophoresis showing exon 31 skipping using antisense molecules H31D(+01−25) and H31D(+03−22); and a “cocktail” of antisense molecules directed at exon 31.

FIG. 14 Gel electrophoresis showing exon 33 skipping using antisense molecules H33A(+30+56) and H33A(+64+88) directed at exon 33.

FIG. 15 Gel electrophoresis showing exon 35 skipping using antisense molecules H35A(+141+161), H35A(+116+135), and H35A(+24+43) and a “cocktail of two antisense molecules, directed at exon 35.

FIG. 16 Gel electrophoresis showing exon 36 skipping using antisense molecules H32A(+49+73) and H36A(+26+50) directed at exon 36.

FIG. 17 Gel electrophoresis showing exon 37 skipping using antisense molecules H37A(+82+105) and H37A(+134+157) directed at exon 37.

FIG. 18 Gel electrophoresis showing exon 38 skipping using antisense molecule H38A(+88+112) directed at exon 38.

FIG. 19 Gel electrophoresis showing exon 40 skipping using antisense molecule H40A(−05+17) directed at exon 40.

FIG. 20 Gel electrophoresis showing exon 42 skipping using antisense molecule H42A(−04+23) directed at exon 42.

FIG. 21 Gel electrophoresis showing exon 46 skipping using antisense molecule H46A(+86+115) directed at exon 46

FIG. 22 Gel electrophoresis showing exon 51, exon 52 and exon 53 skipping using various antisense molecules directed at exons 51, 52 and 53, respectively. A “cocktail” of antisense molecules is also shown directed at exon 53.

Brief Description of the Sequence Listings

TABLE 1A Description of 2′-O-methyl phosphorothioate  antisense oligonucleotides that have   been used to date to study induced exon   skipping during the processing of the   dystrophin pre-mRNA. Since these 2′-O-methyl   antisense oligonucleotides are more    RNA-like, U represents uracil. With    other antisense chemistries such as     peptidenucleic acids or morpholinos,these  U bases may be shown as “T”. SEQ ID SEQUENCE NUCLEOTIDE SEQUENCE (5′-3′) 1 H8A(−06+18) GAU AGG UGG UAU CAA CAU  CUG UAA 2 H8A (−03+18) GAU AGG UGG UAU CAA CAU  CUG 3 H8A(−07+18) GAU AGG UGG UAU CAA CAU  CUG UAA G 4 H8A(−06+14) GGU GGU AUC AAC AUC UGU  AA 5 H8A(−10+10) GUA UCA ACA UCU GUA AGC  AC 6 H7A(+45+67) UGC AUG UUC CAG UCG UUG  UGU GG 7 H7A(+02+26) CAC UAU UCC AGU CAA AUA  GGU CUG G 8 H7D(+15−10) AUU UAC CAA CCU UCA GGA  UCG AGU A 9 H7A(−18+03) GGC CUA AAA CAC AUA CAC  AUA 10 C6A(−10+10) CAU UUU UGA CCU ACA UGU  GG 11 C6A(−14+06) UUU GAC CUA CAU GUG GAA  AG 12 C6A(−14+12) UAC AUU UUU GAC CUA CAU  GUG GAA AG 13 C6A(−13+09) AUU UUU GAC CUA CAU GGG  AAA G 14 CH6A(+69+91) UAC GAG UUG AUU GUC GGA  CCC AG 15 C6D(+12−13) GUG GUC UCC UUA CCU AUG  ACU GUG G 16 C6D(+06−11) GGU CUC CUU ACC UAU GA 17 H6D(+04−21) UGU CUC AGU AAU CUU CUU  ACC UAU 18 H6D(+18−04) UCU UAC CUA UGA CUA UGG  AUG AGA 19 H4A(+13+32) GCA UGA ACU CUU GUG GAU  CC 20 H4D(+04−16) CCA GGG UAC UAC UUA CAU  UA 21 H4D(−24−44) AUC GUG UGU CAC AGC AUC  CAG 22 H4A(+11+40) UGU UCA GGG CAU GAA CUC  UUG UGG AUC CUU 23 H3A(+30+60) UAG GAG GCG CCU CCC AUC  CUG UAG GUC ACU G 24 H3A(+35+65) AGG UCU AGG AGG CGC CUC  CCA UCC UGU AGG U 25 H3A(+30+54) GCG CCU CCC AUC CUG UAG  GUC ACU G 26 H3D(+46−21) CUU CGA GGA GGU CUA GGA  GGC GCC UC 27 H3A(+30+50) CUC CCA UCC UGU AGG UCA  CUG 28 H3D(+19−03) UAC CAG UUU UUG CCC UGU  CAG G 29 H3A(−06+20) UCA AUA UGC UGC UUC CCA  AAC UGA AA 30 H3A(+37+61) CUA GGA GGC GCC UCC CAU  CCU GUA G 31 H5A(+20+50) UUA UGA UUU CCA UCU ACG  AUG UCA GUA CUU C 32 H5D(+25−05) CUU ACC UGC CAG UGG AGG  AUU AUA UUC CAA A 33 H5D(+10−15) CAU CAG GAU UCU UAC CUG  CCA GUG G 34 H5A(+10+34) CGA UGU CAG UAC UUC CAA  UAU UCA C 35 H5D(−04−21) ACC AUU CAU CAG GAU UCU 36 H5D(+16−02) ACC UGC CAG UGG AGG AUU 37 H5A(−07+20) CCA AUA UUC ACU AAA UCA  ACC UGU UAA 38 H5D(+18−12) CAG GAU UCU UAC CUG CCA  GUG GAG GAU UAU 39 H5A(+05+35) ACG AUG UCA GUA CUU CCA  AUA UUC ACU AAA U 40 H5A(+15+45) AUU UCC AUC UAC GAU GUC  AGU ACU UCC AAU A 41 H10A(−05+16) CAG GAG CUU CCA AAU GCU  GCA 42 H10A(−05+24) CUU GUC UUC AGG AGC UUC  CAA AUG CUG CA 43 H10A(+98+119) UCC UCA GCA GAA AGA AGC  CAC G 44 H10A(+130+149) UUA GAA AUC UCU CCU UGU  GC 45 H10A(−33−14) UAA AUU GGG UGU UAC ACA  AU 46 H11D(+26+49) CCC UGA GGC AUU CCC AUC  UUG AAU 47 H11D(+11−09) AGG ACU UAC UUG CUU UGU  UU 48 H11A(+118+140) CUU GAA UUU AGG AGA UUC  AUC UG 49 H11A(+75+97) CAU CUU CUG AUA AUU UUC  CUG UU 50 H12A(+52+75) UCU UCU GUU UUU GUU AGC  CAG UCA 51 H12A(−10+10) UCU AUG UAA ACU GAA AAU  UU 52 H12A(+11+30) UUC UGG AGA UCC AUU AAA  AC 53 H13A(+77+100) CAG CAG UUG CGU GAU CUC  CAC UAG 54 H13A(+55+75) UUC AUC AAC UAC CAC CAC  CAU 55 H13D(+06−19) CUA AGC AAA AUA AUC UGA  CCU UAA G 56 H14A(+37+64) CUU GUA AAA GAA CCC AGC  GGU CUU CUG U 57 H14A(+14+35) CAU CUA CAG AUG UUU GCC  CAU C 58 H14A(+51+73) GAA GGA UGU CUU GUA AAA  GAA CC 59 H14D(−02+18) ACC UGU UCU UCA GUA AGA  CG 60 H14D(+14−10) CAU GAC ACA CCU GUU CUU  CAG UAA 61 H14A(+61+80) CAU UUG AGA AGG AUG UCU  UG 62 H14A(−12+12) AUC UCC CAA UAC CUG GAG  AAG AGA 63 H15A(−12+19) GCC AUG CAC UAA AAA GGC  ACU GCA AGA CAU U 64 H15A(+48+71) UCU UUA AAG CCA GUU GUG  UGA AUC 65 H15A(+08+28) UUU CUG AAA GCC AUG CAC  UAA 66 H15D(+17−08) GUA CAU ACG GCC AGU UUU  UGA AGA C 67 H16A(−12+19) CUA GAU CCG CUU UUA AAA  CCU GUU AAA ACA A 68 H16A(−06+25) UCU UUU CUA GAU CCG CUU  UUA AAA CCU GUU A 69 H16A(−06+19) CUA GAU CCG CUU UUA AAA  CCU GUU A 70 H16A(+87+109) CCG UCU UCU GGG UCA CUG  ACU UA 71 H16A(−07+19) CUA GAU CCG CUU UUA AAA  CCU GUU AA 72 H16A(−07+13) CCG CUU UUA AAA CCU GUU  AA 73 H16A(+12+37) UGG AUU GCU UUU UCU UUU  CUA GAU CC 74 H16A(+92+116) CAU GCU UCC GUC UUC UGG  GUC ACU G 75 H16A(+45+67) G AUC UUG UUU GAG UGA  AUA CAG U 76 H16A(+105+126) GUU AUC CAG CCA UGC UUC  CGU C 77 H16D(+05−20) UGA UAA UUG GUA UCA CUA  ACC UGU G 78 H16D(+12−11) GUA UCA CUA ACC UGU GCU  GUA C 79 H19A(+35+53) CAG CAG UAG UUG UCA UCU  GC 80 H19A(+35+65) GCC UGA GCU GAU CUG CUG  GCA UCU UGC AGU U 81 H20A(+44+71) CUG GCA GAA UUC GAU CCA  CCG GCU GUU C 82 H20A(+149+170) CAG CAG UAG UUG UCA UCU  GCU C 83 H20A(+185+203) UGA UGG GGU GGU GGG UUG  G 84 H20A(−08+17) AUC UGC AUU AAC ACC CUC  UAG AAA G 85 H20A(+30+53) CCG GCU GUU CAG UUG UUC  UGA GGC 86 H20A(−11+17) AUC UGC AUU AAC ACC CUC  UAG AAA GAA A 87 H20D(+08−20) GAA GGA GAA GAG AUU CUU  ACC UUA CAA A 88 H20A(+44+63) AUU CGA UCC ACC GGC UGU  UC 89 H20A(+149+168 CUG CUG GCA UCU UGC AGU  U 90 H21A(−06+16) GCC GGU UGA CUU CAU CCU  GUG C 91 H21A(+85+106) CUG CAU CCA GGA ACA UGG  GUC C 92 H21A(+85+108) GUC UGC AUC CAG GAA CAU  GGG UC 93 H21A(+08+31) GUU GAA GAU CUG AUA GCC  GGU UGA 94 H21D(+18−07) UAC UUA CUG UCU GUA GCU  CUU UCU 95 H22A(+22+45) CAC UCA UGG UCU CCU GAU  AGC GCA 96 H22A(+125+106) CUG CAA UUC CCC GAG UCU  CUG C 97 H22A(+47+69) ACU GCU GGA CCC AUG UCC  UGA UG 98 H22A(+80+101) CUA AGU UGA GGU AUG GAG  AGU 99 H22D(+13−11) UAU UCA CAG ACC UGC AAU  UCC CC 100 H23A(+34+59) ACA GUG GUG CUG AGA UAG  UAU AGG CC 101 H23A(+18+39) UAG GCC ACU UUG UUG CUC  UUG C 102 H23A(+72+90) UUC AGA GGG CGC UUU CUU  C 103 H24A(+48+70) GGG CAG GCC AUU CCU CCU  UCA GA 104 H24A(−02+22) UCU UCA GGG UUU GUA UGU  GAU UCU 105 H25A(+9+36) CTG GGC UGA AUU GUC UGA AUA UCA CUG 106 H25A(+131+156) CUG UUG GCA CAU GUG AUC  CCA CUG AG 107 H25D(+16−08) GUC UAU ACC UGU UGG CAC  AUG UGA 108 H26A(+132+156) UGC UUU CUG UAA UUC AUC  UGG AGU U 109 H26A(−07+19) CCU CCU UUC UGG CAU AGA  CCU UCC AC 110 H26A(+68+92) UGU GUC AUC CAU UCG UGC  AUC UCU G 111 H27A(+82+106) UUA AGG CCU CUU GUG CUA  CAG GUG G 112 H27A(−4+19) GGG CCU CUU CUU UAG CUC  UCU GA 113 H27D(+19−03) GAC UUC CAA AGU CUU GCA  UUU C 114 H28A(−05+19) GCC AAC AUG CCC AAA CUU  CCU AAG 115 H28A(+99+124) CAG AGA UUU CCU CAG CUC  CGC CAG GA 116 H28D(+16−05) CUU ACA UCU AGC ACC UCA  GAG 117 H29A(+57+81) UCC GCC AUC UGU UAG GGU  CUG UGC C 118 H29A(+18+42) AUU UGG GUU AUC CUC UGA  AUG UCG C 119 H29D(+17−05) CAU ACC UCU UCA UGU AGU  UCU C 120 H30A(+122+147) CAU UUG AGC UGC GUC CAC  CUU GUC UG 121 H30A(+25+50) UCC UGG GCA GAC UGG AUG  CUC UGU UC 122 H30D(+19−04) UUG CCU GGG CUU CCU GAG  GCA UU 123 H31D(+06−18) UUC UGA AAU AAC AUA UAC  CUG UGC 124 H31D(+03−22) UAG UUU CUG AAA UAA CAU  AUA CCU G 125 H31A(+05+25) GAC UUG UCA AAU CAG AUU  GGA 126 H31D(+04−20) GUU UCU GAA AUA ACA UAU  ACC UGU 127 H32D(+04−16) CAC CAG AAA UAC AUA CCA CA 128 H32A(+151+170) CAA UGA UUU AGC UGU GAC  UG 129 H32A(+10+32) CGA AAC UUC AUG GAG ACA  UCU UG 130 H32A(+49+73) CUU GUA GAC GCU GCU CAA  AAU UGG C 131 H33D(+09−11) CAU GCA CAC ACC UUU GCU  CC 132 H33A(+53+76) UCU GUA CAA UCU GAC GUC  CAG UCU 133 H33A(+30+56) GUC UUU AUC ACC AUU UCC  ACU UCA GAC 134 H33A(+64+88) CCG UCU GCU UUU UCU GUA  CAA UCU G 135 H34A(+83+104) UCC AUA UCU GUA GCU GCC  AGC C 136 H34A(+143+165) CCA GGC AAC UUC AGA AUC  CAA AU 137 H34A(−20+10) UUU CUG UUA CCU GAA AAG  AAU UAU AAU GAA 138 H34A(+46+70) CAU UCA UUU CCU UUC GCA  UCU UAC G 139 H34A(+95+120) UGA UCU CUU UGU CAA UUC  CAU AUC UG 140 H34D(+10−20) UUC AGU GAU AUA GGU UUU  ACC UUU CCCCAG 141 H34A(+72+96) CUG UAG CUG CCA GCC AUU  CUG UCA AG 142 H35A(+141+161) UCU UCU GCU CGG GAG GUG  ACA 143 H35A(+116+135) CCA GUU ACU AUU CAG AAG  AC 144 H35A(+24+43) UCU UCA GGU GCA CCU UCU  GU 145 H36A(+26+50) UGU GAU GUG GUC CAC AUU  CUG GUC A 146 H36A(−02+18) CCA UGU GUU UCU GGU AUU  CC 147 H37A(+26+50) CGU GUA GAG UCC ACC UUU  GGG CGU A 148 H37A(+82+105) UAC UAA UUU CCU GCA GUG  GUC ACC 149 H37A(+134+157) UUC UGU GUG AAA UGG CUG  CAA AUC 150 H38A(−01+19) CCU UCA AAG GAA UGG AGG  CC 151 H38A(+59+83) UGC UGA AUU UCA GCC UCC AGU GGU U 152 H38A(+88+112) UGA AGU CUU CCU CUU UCA  GAU UCA C 153 H39A(+62+85) CUG GCU UUC UCU CAU CUG  UGA UUC 154 H39A(+39+58) GUU GUA AGU UGU CUC CUC  UU 155 H39A(+102+121) UUG UCU GUA ACA GCU GCU  GU 156 H39D(+10−10) GCU CUA AUA CCU UGA GAG  CA 157 H40A(−05+17) CUU UGA GAC CUC AAA UCC  UGU U 158 H40A(+129+153) CUU UAU UUU CCU UUC AUC  UCU GGG C 159 H42A(−04+23) AUC GUU UCU UCA CGG ACA  GUG UGC UGG 160 H42A(+86+109) GGG CUU GUG AGA CAU GAG  UGA UUU 161 H42D(+19−02) A CCU UCA GAG GAC UCC  UCU UGC 162 H43D(+10−15) UAU GUG UUA CCU ACC CUU  GUC GGU C 163 H43A(+101+120) GGA GAG AGC UUC CUG UAG  CU 164 H43A(+78+100) UCA CCC UUU CCA CAG GCG  UUG CA 165 H44A(+85+104) UUU GUG UCU UUC UGA GAA  AC 166 H44D(+10−10) AAA GAC UUA CCU UAA GAU  AC 167 H44A(−06+14) AUC UGU CAA AUC GCC UGC  AG 168 H46D(+16−04) UUA CCU UGA CUU GCU CAA  GC 169 H46A(+90+109) UCC AGG UUC AAG UGG GAU  AC 170 H47A(+76+100) GCU CUU CUG GGC UUA UGG  GAG CAC U 171 H47D(+25−02) ACC UUU AUC CAC UGG AGA  UUU GUC UGC 172 H47A(−9+12) UUC CAC CAG UAA CUG AAA  CAG 173 H50A(+02+30) CCA CUC AGA GCU CAG AUC  UUC UAA CUU CC 174 H50A(+07+33) CUU CCA CUC AGA GCU CAG  AUC UUC UAA 175 H50D(+07−18) GGG AUC CAG UAU ACU UAC  AGG CUC C 176 H51A(−01+25) ACC AGA GUA ACA GUC UGA  GUA GGA GC 177 H51D(+16−07) CUC AUA CCU UCU GCU UGA  UGA UC 178 H51A(+111+134) UUC UGU CCA AGC CCG GUU  GAA AUC 179 H51A(+61+90) ACA UCA AGG AAG AUG GCA  UUU CUA GUU UGG 180 H51A(+66+90) ACA UCA AGG AAG AUG GCA  UUU CUA G 181 H51A(+66+95) CUC CAA CAU CAA GGA AGA  UGG CAU UUC UAG 182 H51D(+08−17) AUC AUU UUU UCU CAU ACC  UUC UGC U 183 H51A/D(+08−17) AUC AUU UUU UCU CAU ACC  & (−15+) UUC UGC UAG GAG CUA AAA 184 H51A(+175+195) CAC CCA CCA UCA CCC UCU  GUG 185 H51A(+199+220) AUC AUC UCG UUG AUA UCC  UCA A 186 H52A(−07+14) UCC UGC AUU GUU GCC UGU  AAG 187 H52A(+12+41) UCC AAC UGG GGA CGC CUC  UGU UCC AAA UCC 188 H52A(+17+37) ACU GGG GAC GCC UCU GUU  CCA 189 H52A(+93+112) CCG UAA UGA UUG UUC UAG  CC 190 H52D(+05−15) UGU UAA AAA ACU UAC UUC  GA 191 H53A(+45+69) CAU UCA ACU GUU GCC UCC  GGU UCU G 192 H53A(+39+62) CUG UUG CCU CCG GUU CUG  AAG GUG 193 H53A(+39+69) CAU UCA ACU GUU GCC UCC  GGU UCU GAA GGU G 194 H53D(+14−07) UAC UAA CCU UGG UUU CUG  UGA 195 H53A(+23+47) CUG AAG GUG UUC UUG UAC  UUC AUC C 196 H53A(+150+176) UGU AUA GGG ACC CUC CUU  CCA UGA CUC 197 H53D(+20−05) CUA ACC UUG GUU UCU GUG  AUU UUC U 198 H53D(+09−18) GGU AUC UUU GAU ACU AAC  CUU GGU UUC 199 H53A(−12+10) AUU CUU UCA ACU AGA AUA  AAA G 200 H53A(−07+18) GAU UCU GAA UUC UUU CAA  CUA GAA U 201 H53A(+07+26) AUC CCA CUG AUU CUG AAU  UC 202 H53A(+124+145) UUG GCU CUG GCC UGU CCU  AAG A 203 H46A(+86+115) CUC UUU UCC AGG UUC AAG  UGG GAU ACU AGC 204 H46A(+107+137) CAA GCU UUU CUU UUA GUU  GCU GCU CUU UUC C 205 H46A(−10+20) UAU UCU UUU GUU CUU CUA  GCC UGG AGA AAG 206 H46A(+50+77) CUG CUU CCU CCA ACC AUA  AAA CAA AUU C 207 H45A(−06+20) CCA AUG CCA UCC UGG AGU  UCC UGU AA 208 H45A(+91+110) UCC UGU AGA AUA CUG GCA  UC 209 H45A(+125+151) UGC AGA CCU CCU GCC ACC  GCA GAU UCA 210 H45D(+16−04) CUA CCU CUU UUU UCU GUC  UG 211 H45A(+71+90) UGU UUU UGA GGA UUG CUG  AA

TABLE 1B Description of a cocktail of 2′-O-methyl  phosphorothioate antisense oligonucleotides  that have been used to date to study  induced exon skipping during the processing of the dystrophin pre-mRNA. SEQ ID SEQUENCE NUCLEOTIDE SEQUENCE (5′-3′) 81 H20A(+44+71) CUG GCA GAA UUC GAU CCA  CCG GCU GUU C 82 H20A(+149+170) CAG CAG UAG UUG UCA UCU  GCU C 79 H19A(+35+65) GCC UGA GCU GAU CUG CUG  GCA UCU UGC AGU U 81 H20A(+44+71) CUG GCA GAA UUC GAU CCA  CCG GCU GUU C 82 H20A(+149+170) CAG CAG UAG UUG UCA UCU  GCU C 194 H53D(+14−07) UAC UAA CCU UGG UUU CUG  UGA 195 H53A(+23+47) CTG AAG GUG UUC UUG UAC  UUC AUC C 196 H53A(+150+175) UGU AUA GGG ACC CUC CUU  CCA UGA CUC

TABLE 1C Description of a “weasel” of 2′-O-methyl phosphorothioate antisense oligonucleotides  that have been used to date to study  induced exon skipping during the processing  of the dystrophin pre-mRNA. SEQ ID SEQUENCE NUCELOTIDE SEQUENCE (5′-3′)  80 H20A(+44+71)- CUG GCA GAA UUC GAU CCA  CCG GCU GUU C-  82 H20A(+149+170) CAG CAG UAG UUG UCA UCU  GCU C  81 H19A(+35+53)- GCC UGA GCU GAU CUG   CUG GCA UCU UGC AGU U  88 H20A(+44+63)- -AUU CGA UCU ACC GGC   UGU UC-  89 H20A(+149+168) AA CUG CUG GCA UCU UGC  AGU U  80 H19A(+35+53)- GCC UGA GCU GAU CUG CUG  GCA UCU UGC AGU U  88 H20A(+44+63) -AUU CGA UCU ACC GGC  UGU UC-  80 H19A(+35+53)- GCC UGA GCU GAU CUG CUG  GCA UCU UGC   89 H20A(+149+168) AGU U -AA CUG CUG GCA UCU UGC  AGU U 138 H34A(+46+70)- CAU UCA UUU CCU UUC GCA  UCU UAC G- 139 H34A(+94+120) UGA UCU CUU UGU CAA UUC  CAU AUC UG 124 H31D(+03−22)- UAG UUU CUG AAA UAA CAU  UU- AUA CCU G- UU- 144 H35A(+24+43) UCU UCA GGU GCA CCU UCU  GU 195 H53A(+23+47)- CUG AAG GUG UUC UUG UAC  AA- UUC AUC C- 196 H53A(+150+ UGU AUA GGG ACC CUC CUU   175)-AA- CCA UGA CUC-AA- 194 H53D(+14−07) UAC UAA CCU UGG UUU CUG  — Aimed at exons UGA CAG CAG UAG UUG UCA 19/20/20 UCU GCU CAA CUG GCA GAA UUC GAU CCA CCG GCU GUU CAA GCC UGA GCU GAU CUG CUC GCA UCU UGC AGU

DETAILED DESCRIPTION OF THE INVENTION General

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variation and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

Sequence identity numbers (SEQ ID NO:) containing nucleotide and amino acid sequence information included in this specification are collected at the end of the description and have been prepared using the programme PatentIn Version 3.0. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, etc.). The length, type of sequence and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (e.g. <400>1, <400>2, etc.).

An antisense molecules nomenclature system was proposed and published to distinguish between the different antisense molecules (see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature became especially relevant when testing several slightly different antisense molecules, all directed at the same target region, as shown below:

-   -   H # A/D (x: y).         The first letter designates the species (e.g. H: human, M:         murine, C: canine)         “#” designates target dystrophin exon number.         “A/D” indicates acceptor or donor splice site at the beginning         and end of the exon, respectively.         (x y) represents the annealing coordinates where “−” or “+”         indicate intronic or exonic sequences respectively. As an         example, A(−6+18) would indicate the last 6 bases of the intron         preceding the target exon and the first 18 bases of the target         exon. The closest splice site would be the acceptor so these         coordinates would be preceded with an “A”. Describing annealing         coordinates at the donor splice site could be D(+2−18) where the         last 2 exonic bases and the first 18 intronic bases correspond         to the annealing site of the antisense molecule. Entirely exonic         annealing coordinates that would be represented by A(+65+85),         that is the site between the 65^(th) and 85^(th) nucleotide from         the start of that exon.

The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.

As used necessarily herein the term “derived” and “derived from” shall be taken to indicate that a specific integer may be obtained from a particular source albeit not directly from that source.

Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

DESCRIPTION OF THE PREFERRED EMBODIMENT

When antisense molecule(s) are targeted to nucleotide sequences involved in splicing in exons within pre-mRNA sequences, normal splicing of the exon may be inhibited causing the splicing machinery to by-pass the entire mutated exon from the mature mRNA. The concept of antisense oligonucleotide induced exon skipping is shown in FIG. 2. In many genes, deletion of an entire exon would lead to the production of a non-functional protein through the loss of important functional domains or the disruption of the reading frame. In some proteins, however, it is possible to shorten the protein by deleting one or more exons, without disrupting the reading frame, from within the protein without seriously altering the biological activity of the protein. Typically, such proteins have a structural role and or possess functional domains at their ends. The present invention describes antisense molecules capable of binding to specified dystrophin pre-mRNA targets and re-directing processing of that gene.

Antisense Molecules

According to a first aspect of the invention, there is provided antisense molecules capable of binding to a selected target to induce exon skipping. To induce exon skipping in exons of the Dystrophin gene transcript, the antisense molecules are preferably selected from the group of compounds shown in Table 1A. There is also provided a combination or “cocktail” of two or more antisense oligonucleotides capable of binding to a selected target to induce exon skipping. To induce exon skipping in exons of the Dystrophin gene transcript, the antisense molecules in a “cocktail” are preferably selected from the group of compounds shown in Table 1B. Alternatively, exon skipping may be induced by antisense oligonucleotides joined together “weasels” preferably selected from the group of compounds shown in Table 1C.

Designing antisense molecules to completely mask consensus splice sites may not necessarily generate any skipping of the targeted exon. Furthermore, the inventors have discovered that size or length of the antisense oligonucleotide itself is not always a primary factor when designing antisense molecules. With some targets such as exon 19, antisense oligonucleotides as short as 12 bases were able to induce exon skipping, albeit not as efficiently as longer (20-31 bases) oligonucleotides. In some other targets, such as murine dystrophin exon 23, antisense oligonucleotides only 17 residues long were able to induce more efficient skipping than another overlapping compound of 25 nucleotides.

The inventors have also discovered that there does not appear to be any standard motif that can be blocked or masked by antisense molecules to redirect splicing. In some exons, such as mouse dystrophin exon 23, the donor splice site was the most amenable to target to re-direct skipping of that exon. It should be noted that designing and testing a series of exon 23 specific antisense molecules to anneal to overlapping regions of the donor splice site showed considerable variation in the efficacy of induced exon skipping. As reported in Mann et al., (2002) there was a significant variation in the efficiency of bypassing the nonsense mutation depending upon antisense oligonucleotide annealing (“Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy”. J Gen Med 4: 644-654). Targeting the acceptor site of exon 23 or several internal domains was not found to induce any consistent exon 23 skipping.

In other exons targeted for removal, masking the donor splice site did not induce any exon skipping. However, by directing antisense molecules to the acceptor splice site (human exon 8 as discussed below), strong and sustained exon skipping was induced. It should be noted that removal of human exon 8 was tightly linked with the co-removal of exon 9. There is no strong sequence homology between the exon 8 antisense oligonucleotides and corresponding regions of exon 9 so it does not appear to be a matter of cross reaction. Rather the splicing of these two exons is inextricably linked. This is not an isolated instance as the same effect is observed in canine cells where targeting exon 8 for removal also resulted in the skipping of exon 9. Targeting exon 23 for removal in the mouse dystrophin pre-mRNA also results in the frequent removal of exon 22 as well. This effect occurs in a dose dependent manner and also indicates close coordinated processing of 2 adjacent exons.

In other targeted exons, antisense molecules directed at the donor or acceptor splice sites did not induce exon skipping while annealing antisense molecules to intra-exonic regions (i.e. exon splicing enhancers within human dystrophin exon 6) was most efficient at inducing exon skipping. Some exons, both mouse and human exon 19 for example, are readily skipped by targeting antisense molecules to a variety of motifs. That is, targeted exon skipping is induced after using antisense oligonucleotides to mask donor and acceptor splice sites or exon splicing enhancers.

To identify and select antisense oligonucleotides suitable for use in the modulation of exon skipping, a nucleic acid sequence whose function is to be modulated must first be identified. This may be, for example, a gene (or mRNA transcribed form the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. Within the context of the present invention, preferred target site(s) are those involved in mRNA splicing (i.e. splice donor sites, splice acceptor sites, or exonic splicing enhancer elements). Splicing branch points and exon recognition sequences or splice enhancers are also potential target sites for modulation of mRNA splicing.

Preferably, the present invention aims to provide antisense molecules capable of binding to a selected target in the dystrophin pre-mRNA to induce efficient and consistent exon skipping. Duchenne muscular dystrophy arises from mutations that preclude the synthesis of a functional dystrophin gene product. These Duchenne muscular dystrophy gene defects are typically nonsense mutations or genomic rearrangements such as deletions, duplications or micro-deletions or insertions that disrupt the reading frame. As the human dystrophin gene is a large and complex gene with the 79 exons being spliced together to generate a mature mRNA with an open reading frame of approximately 11,000 bases, there are many positions where these mutations can occur. Consequently, a comprehensive antisense oligonucleotide based therapy to address many of the different disease-causing mutations in the dystrophin gene will require that many exons can be targeted for removal during the splicing process.

Within the context of the present invention, preferred target site(s) are those involved in mRNA splicing (i.e. splice donor sites, splice acceptor sites or exonic splicing enhancer elements). Splicing branch points and exon recognition sequences or splice enhancers are also potential target sites for modulation of mRNA splicing.

The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridisable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense molecule need not be 100% complementary to that of its target sequence to be specifically hybridisable. An antisense molecule is specifically hybridisable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

While the above method may be used to select antisense molecules capable of deleting any exon from within a protein that is capable of being shortened without affecting its biological function, the exon deletion should not lead to a reading frame shift in the shortened transcribed mRNA. Thus, if in a linear sequence of three exons the end of the first exon encodes two of three nucleotides in a codon and the next exon is deleted then the third exon in the linear sequence must start with a single nucleotide that is capable of completing the nucleotide triplet for a codon. If the third exon does not commence with a single nucleotide there will be a reading frame shift that would lead to the generation of truncated or a non-functional protein.

It will be appreciated that the codon arrangements at the end of exons in structural proteins may not always break at the end of a codon, consequently there may be a need to delete more than one exon from the pre-mRNA to ensure in-frame reading of the mRNA. In such circumstances, a plurality of antisense oligonucleotides may need to be selected by the method of the invention wherein each is directed to a different region responsible for inducing splicing in the exons that are to be deleted.

The length of an antisense molecule may vary so long as it is capable of binding selectively to the intended location within the pre-mRNA molecule. The length of such sequences can be determined in accordance with selection procedures described herein. Generally, the antisense molecule will be from about 10 nucleotides in length up to about 50 nucleotides in length. It will be appreciated however that any length of nucleotides within this range may be used in the method. Preferably, the length of the antisense molecule is between 17 to 30 nucleotides in length.

In order to determine which exons can be connected in a dystrophin gene, reference should be made to an exon boundary map. Connection of one exon with another is based on the exons possessing the same number at the 3′ border as is present at the 5′ border of the exon to which it is being connected. Therefore, if exon 7 were deleted, exon 6 must connect to either exons 12 or 18 to maintain the reading frame. Thus, antisense oligonucleotides would need to be selected which redirected splicing for exons 7 to 11 in the first instance or exons 7 to 17 in the second instance. Another and somewhat simpler approach to restore the reading frame around an exon 7 deletion would be to remove the two flanking exons. Induction of exons 6 and 8 skipping should result in an in-frame transcript with the splicing of exons 5 to 9. In practise however, targeting exon 8 for removal from the pre-mRNA results in the co-removal of exon 9 so the resultant transcript would have exon 5 joined to exon 10. The inclusion or exclusion of exon 9 does not alter the reading frame. Once the antisense molecules to be tested have been identified, they are prepared according to standard techniques known in the art. The most common method for producing antisense molecules is the methylation of the 2′ hydroxyribose position and the incorporation of a phosphorothioate backbone produces molecules that superficially resemble RNA but that are much more resistant to nuclease degradation.

To avoid degradation of pre-mRNA during duplex formation with the antisense molecules, the antisense molecules used in the method may be adapted to minimise or prevent cleavage by endogenous RNase H. This property is highly preferred as the treatment of the RNA with the unmethylated oligonucleotides either intracellularly or in crude extracts that contain RNase H leads to degradation of the pre-mRNA: antisense oligonucleotide duplexes. Any form of modified antisense molecules that is capable of by-passing or not inducing such degradation may be used in the present method. An example of antisense molecules which when duplexed with RNA are not cleaved by cellular RNase H is 2′-O-methyl derivatives. 2′-O-methyl-oligoribonucleotides are very stable in a cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo- counterparts.

Antisense molecules that do not activate RNase H can be made in accordance with known techniques (see, e.g., U.S. Pat. No. 5,149,797). Such antisense molecules, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification which sterically hinders or prevents binding of RNase H to a duplex molecule containing the oligonucleotide as one member thereof, which structural modification does not substantially hinder or disrupt duplex formation. Because the portions of the oligonucleotide involved in duplex formation are substantially different from those portions involved in RNase H binding thereto, numerous antisense molecules that do not activate RNase H are available. For example, such antisense molecules may be oligonucleotides wherein at least one, or all, of the inter-nucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues may be modified as described. In another non-limiting example, such antisense molecules are molecules wherein at least one, or all, of the nucleotides contain a 2′ lower alkyl moiety (e.g., C₁-C₄, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other one of the nucleotides may be modified as described.

While antisense oligonucleotides are a preferred form of the antisense molecules, the present invention comprehends other oligomeric antisense molecules, including but not limited to oligonucleotide mimetics such as are described below.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural inter-nucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their inter-nucleoside backbone can also be considered to be oligonucleosides.

In other preferred oligonucleotide mimetics, both the sugar and the inter-nucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleo-bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Certain nucleo-bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds that are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense molecules, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid.

Methods of Manufacturing Antisense Molecules

The antisense molecules used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesising oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.

Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. In one such automated embodiment, diethyl-phosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al., (1981) Tetrahedron Letters, 22:1859-1862.

The antisense molecules of the invention are synthesised in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The molecules of the invention may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.

Therapeutic Agents

The present invention also can be used as a prophylactic or therapeutic, which may be utilised for the purpose of treatment of a genetic disease.

Accordingly, in one embodiment the present invention provides antisense molecules that bind to a selected target in the dystrophin pre-mRNA to induce efficient and consistent exon skipping described herein in a therapeutically effective amount admixed with a pharmaceutically acceptable carrier, diluent, or excipient.

The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similarly untoward reaction, such as gastric upset and the like, when administered to a patient. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa., (1990).

In a more specific form of the invention there are provided pharmaceutical compositions comprising therapeutically effective amounts of an antisense molecule together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The material may be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 that are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilised form.

It will be appreciated that pharmaceutical compositions provided according to the present invention may be administered by any means known in the art. Preferably, the pharmaceutical compositions for administration are administered by injection, orally, or by the pulmonary, or nasal route. The antisense molecules are more preferably delivered by intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous routes of administration.

Antisense Molecule Based Therapy

Also addressed by the present invention is the use of antisense molecules of the present invention, for manufacture of a medicament for modulation of a genetic disease.

The delivery of a therapeutically useful amount of antisense molecules may be achieved by methods previously published. For example, intracellular delivery of the antisense molecule may be via a composition comprising an admixture of the antisense molecule and an effective amount of a block copolymer. An example of this method is described in US patent application US 20040248833.

Other methods of delivery of antisense molecules to the nucleus are described in Mann C J et al., (2001) [“Antisense-induced exon skipping and the synthesis of dystrophin in the mdx mouse”. Proc. Natl. Acad. Science, 98(1) 42-47] and in Gebski et al., (2003). Human Molecular Genetics, 12(15): 1801-1811.

A method for introducing a nucleic acid molecule into a cell by way of an expression vector either as naked DNA or complexed to lipid carriers, is described in U.S. Pat. No. 6,806,084.

It may be desirable to deliver the antisense molecule in a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations.

Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. These formulations may have net cationic, anionic or neutral charge characteristics and are useful characteristics with in vitro, in vivo and ex vivo delivery methods. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 .PHI.m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, and DNA can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981).

In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the antisense molecule of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Alternatively, the antisense construct may be combined with other pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.

The routes of administration described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and any dosage for any particular animal and condition. Multiple approaches for introducing functional new genetic material into cells, both in vitro and in vivo have been attempted (Friedmann (1989) Science, 244:1275-1280).

These approaches include integration of the gene to be expressed into modified retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer Research 51(18), suppl.: 5074S-5079S); integration into non-retrovirus vectors (Rosenfeld, et al. (1992) Cell, 68:143-155; Rosenfeld, et al. (1991) Science, 252:431-434); or delivery of a transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; Nabel, et al. (1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific, cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA, expression vectors (Nabel et al. (1990), supra); Wolff et al. (1990) Science, 247:1465-1468). Direct injection of transgenes into tissue produces only localized expression (Rosenfeld (1992) supra); Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra). The Brigham et al. group (Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) have reported in vivo transfection only of lungs of mice following either intravenous or intratracheal administration of a DNA liposome complex. An example of a review article of human gene therapy procedures is: Anderson, Science (1992) 256:808-813.

The antisense molecules of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such pro-drugs, and other bioequivalents.

The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, (including by nebulizer, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Kits of the Invention

The invention also provides kits for treatment of a patient with a genetic disease which kit comprises at least an antisense molecule, packaged in a suitable container, together with instructions for its use.

In a preferred embodiment, the kits will contain at least one antisense molecule as shown in Table 1A, or a cocktail of antisense molecules as shown in Table 1B or a “weasel” compound as shown in Table 1C. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

Those of ordinary skill in the field should appreciate that applications of the above method has wide application for identifying antisense molecules suitable for use in the treatment of many other diseases.

EXAMPLES

The following Examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these Examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. The references cited herein are expressly incorporated by reference.

Methods of molecular cloning, immunology and protein chemistry, which are not explicitly described in the following examples, are reported in the literature and are known by those skilled in the art. General texts that described conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, included, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd., Oxford, U.K. (1985); and Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley Intersciences, New York (2002).

Determining Induced Exon Skipping in Human Muscle Cells

Attempts by the inventors to develop a rational approach in antisense molecules design were not completely successful as there did not appear to be a consistent trend that could be applied to all exons. As such, the identification of the most effective and therefore most therapeutic antisense molecules compounds has been the result of empirical studies.

These empirical studies involved the use of computer programs to identify motifs potentially involved in the splicing process. Other computer programs were also used to identify regions of the pre-mRNA which may not have had extensive secondary structure and therefore potential sites for annealing of antisense molecules. Neither of these approaches proved completely reliable in designing antisense oligonucleotides for reliable and efficient induction of exon skipping.

Annealing sites on the human dystrophin pre-mRNA were selected for examination, initially based upon known or predicted motifs or regions involved in splicing. 2OMe antisense oligonucleotides were designed to be complementary to the target sequences under investigation and were synthesised on an Expedite 8909 Nucleic Acid Synthesiser. Upon completion of synthesis, the oligonucleotides were cleaved from the support column and de-protected in ammonium hydroxide before being desalted. The quality of the oligonucleotide synthesis was monitored by the intensity of the trityl signals upon each deprotection step during the synthesis as detected in the synthesis log. The concentration of the antisense oligonucleotide was estimated by measuring the absorbance of a diluted aliquot at 260 nm.

Specified amounts of the antisense molecules were then tested for their ability to induce exon skipping in an in vitro assay, as described below.

Briefly, normal primary myoblast cultures were prepared from human muscle biopsies obtained after informed consent. The cells were propagated and allowed to differentiate into myotubes using standard culturing techniques. The cells were then transfected with the antisense oligonucleotides by delivery of the oligonucleotides to the cells as cationic lipoplexes, mixtures of antisense molecules or cationic liposome preparations.

The cells were then allowed to grow for another 24 hours, after which total RNA was extracted and molecular analysis commenced. Reverse transcriptase amplification (RT-PCR) was undertaken to study the targeted regions of the dystrophin pre-mRNA or induced exonic re-arrangements.

For example, in the testing of an antisense molecule for inducing exon 19 skipping the RT-PCR test scanned several exons to detect involvement of any adjacent exons. For example, when inducing skipping of exon 19, RT-PCR was carried out with primers that amplified across exons 17 and 21. Amplifications of even larger products in this area (i.e. exons 13-26) were also carried out to ensure that there was minimal amplification bias for the shorter induced skipped transcript. Shorter or exon skipped products tend to be amplified more efficiently and may bias the estimated of the normal and induced transcript.

The sizes of the amplification reaction products were estimated on an agarose gel and compared against appropriate size standards. The final confirmation of identity of these products was carried out by direct DNA sequencing to establish that the correct or expected exon junctions have been maintained.

Once efficient exon skipping had been induced with one antisense molecule, subsequent overlapping antisense molecules may be synthesized and then evaluated in the assay as described above. Our definition of an efficient antisense molecule is one that induces strong and sustained exon skipping at transfection concentrations in the order of 300 nM or less.

Antisense Oligonucleotides Directed at Exon 8

Antisense oligonucleotides directed at exon 8 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 3 shows differing efficiencies of two antisense molecules directed at exon 8 acceptor splice site. H8A(−06+18) [SEQ ID NO:1], which anneals to the last 6 bases of intron 7 and the first 18 bases of exon 8, induces substantial exon 8 and 9 skipping when delivered into cells at a concentration of 20 nM. The shorter antisense molecule, H8A(−06+14) [SEQ ID NO: 4] was only able to induce exon 8 and 9 skipping at 300 nM, a concentration some 15 fold higher than H8A(−06+18), which is the preferred antisense molecule.

This data shows that some particular antisense molecules induce efficient exon skipping while another antisense molecule, which targets a near-by or overlapping region, can be much less efficient. Titration studies show one compound is able to induce targeted exon skipping at 20 nM while the less efficient antisense molecules only induced exon skipping at concentrations of 300 nM and above. Therefore, we have shown that targeting of the antisense molecules to motifs involved in the splicing process plays a crucial role in the overall efficacy of that compound.

Efficacy refers to the ability to induce consistent skipping of a target exon. However, sometimes skipping of the target exons is consistently associated with a flanking exon. That is, we have found that the splicing of some exons is tightly linked. For example, in targeting exon 23 in the mouse model of muscular dystrophy with antisense molecules directed at the donor site of that exon, dystrophin transcripts missing exons 22 and 23 are frequently detected. As another example, when using an antisense molecule directed to exon 8 of the human dystrophin gene, all induced transcripts are missing both exons 8 and 9. Dystrophin transcripts missing only exon 8 are not observed.

Table 2 below discloses antisense molecule sequences that induce exon 8 (and 9) skipping.

TABLE 2 Antisense Ability  Oligo- to nucleotide induce name Sequence skipping H8A(−06+18) 5′-GAU AGG UGG   Very UAU CAA CAU  strong CUG UAA to 20 nM H8A(−03+18) 5′-GAU AGG UGG   Very UAU CAA CAU strong CUG skipping to 40 nM H8A(−07+18) 5′-GAU AGG     Strong UGG UAU CAA  skipping  CAU CUG UAA G to 40 nM H8A(−06+14) 5′-GGU GGU     Skipping AUC AAC AUC to 300 nM UGU AA H8A(−10+10) 5′-GUA UCA     Patchy/ ACA UCU GUA  weak AGC AC skipping to 100 nm

Antisense Oligonucleotides Directed at Exon 7

Antisense oligonucleotides directed at exon 7 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 4 shows the preferred antisense molecule, H7A(+45+67) [SEQ ID NO: 6], and another antisense molecule, H7A(+2+26) [SEQ ID NO: 7], inducing exon 7 skipping. Nested amplification products span exons 3 to 9. Additional products above the induced transcript missing exon 7 arise from amplification from carry-over outer primers from the RT-PCR as well as heteroduplex formation.

Table 3 below discloses antisense molecule sequences for induced exon 7 skipping.

TABLE 3 Antisense Ability Oligo- to  nucleotide induce name Sequence skipping H7A(+45+67) 5′-UGC AUG UUC CAG  Strong  UCG UUG UGU GG skipping to 20 nM H7A(+02+26) 5′-CAC UAU UCC AGU  Weak   CAA AUA GGU CUG G skipping at 100 nM H7D(+15−10) 5′-AUU UAC CAA CCU  Weak   UCA GGA UCG AGU A skipping to 300 nM H7A(−18+03) 5′-GGC CUA AAA CAC  Weak  AUA CAC AUA skipping  to 300 nM

Antisense Oligonucleotides Directed at Exon 6

Antisense oligonucleotides directed at exon 6 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 5 shows an example of two non-preferred antisense molecules inducing very low levels of exon 6 skipping in cultured human cells. Targeting this exon for specific removal was first undertaken during a study of the canine model using the oligonucleotides as listed in Table 4, below. Some of the human specific oligonucleotides were also evaluated, as shown in FIG. 5. In this example, both antisense molecules target the donor splice site and only induced low levels of exon 6 skipping. Both H6D(+4−21) [SEQ ID NO: 17] and H6D(+18−4) [SEQ ID NO: 18] would be regarded as non-preferred antisense molecules.

One antisense oligonucleotide that induced very efficient exon 6 skipping in the canine model, C6A(+69+91) [SEQ ID NO: 14], would anneal perfectly to the corresponding region in human dystrophin exon 6. This compound was evaluated, found to be highly efficient at inducing skipping of that target exon, as shown in FIG. 6 and is regarded as the preferred compound for induced exon 6 skipping. Table 4 below discloses antisense molecule sequences for induced exon 6 skipping.

TABLE 4 Antisense  Ability  Oligo to induce name Sequence skipping C6A(−10+10) 5′ CAU UUU UGA   No  CCU ACA UGU GG skipping C6A(−14+06) 5′ UUU GAC CUA   No  CAU GUG GAA AG skipping C6A(−14+12) 5′ UAC AUU UUU   No  GAC CUA CAU   skipping GUG GAA AG C6A(−13+09) 5′ AUU UUU GAC   No  CUA CAU GGG   skipping AAA G CH6A(+69+91) 5′ UAC GAG UUG   Strong   AUU GUC GGA   skipping CCC AG to 20 nM C6D(+12−13) 5′ GUG GUC UCC   Weak   UUA CCU AUG ACU skipping GUG G at 300 nM C6D(+06−11) 5′ GGU CUC CUU   No  ACC UAU GA skipping H6D(+04−21) 5′ UGU CUC AGU   Weak   AAU CUU CUU ACC skipping UAU to 50 nM H6D(+18−04) 5′ UCU UAC CUA   Very weak  UGA CUA UGG   skipping AUG AGA to 300 nM

Antisense Oligonucleotides Directed at Exon 4

Antisense oligonucleotides directed at exon 4 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 7 shows an example of a preferred antisense molecule inducing skipping of exon 4 skipping in cultured human cells. In this example, one preferred antisense compound, H4A(+13+32) [SEQ ID NO:19], which targeted a presumed exonic splicing enhancer induced efficient exon skipping at a concentration of 20 nM while other non-preferred antisense oligonucleotides failed to induce even low levels of exon 4 skipping. Another preferred antisense molecule inducing skipping of exon 4 was H4A(+111+40) [SEQ ID NO:22], which induced efficient exon skipping at a concentration of 20 nM.

Table 5 below discloses antisense molecule sequences for inducing exon 4 skipping.

TABLE 5 Antisense Ability  Oligo- to nucleotide induce name Sequence skipping H4A(+13+32) 5′ GCA UGA ACU   Skipping  CUU GUG GAU CC to 20 nM H4A(+11+40) 5′UGU UCA GGG   Skipping  CAU GAA CUC   to 20 nM UUG UGG AUC CUU H4D(+04−16) 5′ CCA GGG UAC   No skipping UAC UUA CAU UA H4D(−24−44) 5′ AUC GUG UGU   No skipping CAC AGC AUC CAG

Antisense Oligonucleotides Directed at Exon 3

Antisense oligonucleotides directed at exon 3 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H3A(+30+60) [SEQ ID NO:23] induced substantial exon 3 skipping when delivered into cells at a concentration of 20 nM to 600 nM. The antisense molecule, H3A(+35+65) [SEQ ID NO: 24] induced exon skipping at 300 nM.

Table 6 below discloses antisense molecule sequences that induce exon 3 skipping.

TABLE 6 Antisense Ability - to Oligonucleotide induce name Sequence skipping H3A(+30+60) UAG GAG GCG CCU   Moderate CCC AUC CUG UAG   skipping GUC ACU G to 20 to 600 nM H3A(+35+65) AGG UCU AGG AGG   Working CGC CUC CCA UCC   to UGU AGG U 300 nM H3A(+30+54) GCG CCU CCC AUC   Moderate CUG UAG GUC ACU  100-600 nM G H3D(+46−21) CUU CGA GGA GGU   No skipping CUA GGA GGC GCC  UC H3A(+30+50) CUC CCA UCC UGU   Moderate AGG UCA CUG 20-600 nM H3D(+19−03) UAC CAG UUU UUG   No skipping CCC UGU CAG G H3A(−06+20) UCA AUA UGC UGC   No skipping UUCCCA AAC UGA  AA H3A(+37+61) CUA GGA GGC GCC   No skipping UCC CAU CCU GUA  G

Antisense Oligonucleotides Directed at Exon 5

Antisense oligonucleotides directed at exon 5 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H5A(+20+50) [SEQ ID NO:31] induces substantial exon 5 skipping when delivered into cells at a concentration of 100 nM. Table 7 below shows other antisense molecules tested. The majority of these antisense molecules were not as effective at exon skipping as H5A(+20+50). However, H5A(+15+45) [SEQ ID NO: 40] was able to induce exon 5 skipping at 300 nM.

Table 7 below discloses antisense molecule sequences that induce exon 5 skipping.

TABLE 7 Antisense Ability to Oligonucleotide induce name Sequence skipping H5A(+20+50) UUA UGA UUU CCA UCU  Working to ACG AUG UCA GUA CUU 100 nM C H5D(+25−05) CUU ACC UGC CAG UGG  No skipping AGG AUU AUA UUC CAA A H5D(+10−15) CAU CAG GAU UCU UAC  Inconsistent CUG CCA GUG G at 300 nM H5A(+10+34) CGA UGU CAG UAC UUC  Very weak CAA UAU UCA C H5D(−04−21) ACC AUU CAU CAG GAU  No skipping UCU H5D(+16−02) ACC UGC CAG UGG AGG  No skipping AUU H5A(−07+20) CCA AUA UUC ACU AAA  No skipping UCA ACC UGU UAA H5D(+18−12) CAG GAU UCU UAC CUG  No skipping CCA GUG GAG GAU UAU H5A(+05+35) ACG AUG UCA GUA CUU  No skipping CCA AUA UUC ACU AAA U H5A(+15+45) AUU UCC AUC UAC GAU  Working to GUC AGU ACU UCC AAU 300 nM A

Antisense Oligonucleotides Directed at Exon 10

Antisense oligonucleotides directed at exon 10 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H10A(−05+16) [SEQ ID NO:41] induced substantial exon 10 skipping when delivered into cells. Table 8 below shows other antisense molecules tested. The antisense molecules ability to induce exon skipping was variable. Table 8 below discloses antisense molecule sequences that induce exon 10 skipping.

TABLE 8 Antisense Ability to Oligonucleotide induce name Sequence skipping H10A(−05+16) CAG GAG CUU CCA AAU  Not tested GCU GCA H10A(−05+24) CUU GUC UUC AGG AGC  Not tested UUC CAA AUG CUG CA H10A(+98+119) UCC UCA GCA GAA AGA  Not tested AGC CAC G H10A(+130+149) UUA GAA AUC UCU CCU  No skipping UGU GC H10A(−33−14) UAA AUU GGG UGU UAC  No skipping ACA AU

Antisense Oligonucleotides Directed at Exon 11

Antisense oligonucleotides directed at exon 11 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 8B shows an example of H11A(+75+97) [SEQ ID NO:49] antisense molecule inducing exon 11 skipping in cultured human cells. H11A(+75+97) induced substantial exon 11 skipping when delivered into cells at a concentration of 5 nM. Table 9 below shows other antisense molecules tested. The antisense molecules ability to induce exon skipping was observed at 100 nM.

TABLE 9 Antisense Ability to Oligonucleotide induce name Sequence skipping H11D(+26+49) CCC UGA GGC AUU CCC  Skipping  AUC UUG AAU at 100 nM H11D(+11−09) AGG ACU UAC UUG CUU  Skipping at UGU UU 100 nM H11A(+118+140) CUU GAA UUU AGG AGA  Skipping at UUC AUC UG 100 nM H11A(+75+97) CAU CUU CUG AUA AUU  Skipping at UUC CUG UU 100 nM H11D(+26+49) CCC UGA GGC AUU CCC  Skipping at AUC UUG AAU 5 nM

Antisense Oligonucleotides Directed at Exon 12

Antisense oligonucleotides directed at exon 12 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H12A(+52+75) [SEQ ID NO:50] induced substantial exon 12 skipping when delivered into cells at a concentration of 5 nM, as shown in FIG. 8A. Table 10 below shows other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. The antisense molecules ability to induce exon skipping was variable.

TABLE 10 Antisense Ability to Oligonucleotide induce name Sequence skipping H12A(+52+75) UCU UCU GUU UUU GUU  Skipping at  AGC CAG UCA 5 nM H12A(−10+10) UCU AUG UAA ACU GAA  Skipping at  AAU UU 100 nM H12A(+11+30) UUC UGG AGA UCC AUU  No skipping AAA AC

Antisense Oligonucleotides Directed at Exon 13

Antisense oligonucleotides directed at exon 13 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H13A(+77+100) [SEQ ID NO:53] induced substantial exon 13 skipping when delivered into cells at a concentration of 5 nM. Table 11 below includes two other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These other antisense molecules were unable to induce exon skipping.

TABLE 11 Antisense Ability to Oligonucleotide induce name Sequence skipping H13A(+77+100) CAG CAG UUG CGU GAU Skipping  CUC CAC UAG at 5 nM H13A(+55+75) UUC AUC AAC UAC CAC No  CAC CAU skipping H13D(+06−19) CUA AGC AAA AUA AUC No  UGA CCU UAA G skipping

Antisense Oligonucleotides Directed at Exon 14

Antisense oligonucleotides directed at exon 14 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H14A(+37+64) [SEQ ID NO:56] induced weak exon 14 skipping when delivered into cells at a concentration of 100 nM. Table 12 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. The other antisense molecules were unable to induce exon skipping at any of the concentrations tested.

TABLE 12 Antisense Ability to Oligonucleotide induce name Sequence skipping H14A(+37+64) CUU GUA AAA GAA CCC Skipping AGC GGU CUU CUG U at 100 nM H14A(+14+35) CAU CUA CAG AUG UUU No GCC CAU C skipping H14A(+51+73) GAA GGA UGU CUU GUA No AAA GAA CC skipping H14D(−02+18) ACC UGU UCU UCA GUA No AGA CG skipping H14D(+14−10) CAU GAC ACA CCU GUU No CUU CAG UAA skipping H14A(+61+80) CAU UUG AGA AGG AUG No UCU UG skipping H14A(−12+12) AUC UCC CAA UAC No CUG GAG AAG AGA skipping

Antisense Oligonucleotides Directed at Exon 15

Antisense oligonucleotides directed at exon 15 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H15A(−12+19) [SEQ ID NO:63] and H15A(+48+71) [SEQ ID NO:64] induced substantial exon 15 skipping when delivered into cells at a concentration of 10 Nm, as shown in FIG. 9A. Table 13 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 Nm. These other antisense molecules were unable to induce exon skipping at any of the concentrations tested.

TABLE 13 Antisense Ability to Oligonucleotide induce name Sequence skipping H15A(−12+19) GCC AUG CAC UAA AAA Skipping GGC ACU GCA AGA CAU at 5 Nm U H15A(+48+71) UCU UUA AAG CCA GUU Skipping GUG UGA AUC at 5 Nm H15A(+08+28) UUU CUG AAA GCC AUG No CAC UAA skipping H15A(−12+19) GCC AUG CAC UAA AAA No GGC ACU GCA AGA CAU  skipping U H15D(+17−08) GUA CAU ACG GCC AGU No UUU UGA AGA C skipping

Antisense Oligonucleotides Directed at Exon 16

Antisense oligonucleotides directed at exon 16 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H16A(−12+19) [SEQ ID NO:67] and H16A(−06+25) [SEQ ID NO:68] induced substantial exon 16 skipping when delivered into cells at a concentration of 10 nM, as shown in FIG. 9B. Table 14 below includes other antisense molecules tested. H16A(−06+19) [SEQ ID NO:69] and H16A(+87+109) [SEQ ID NO:70] were tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These two antisense molecules were able to induce exon skipping at 25 nM and 100 nM, respectively. Additional antisense molecules were tested at 100, 200 and 300 nM and did not result in any exon skipping.

TABLE 14 Antisense Ability to Oligonucleotide induce name Sequence skipping H16A(−12+19) CUA GAU CCG CUU UUA Skipping AAA CCU GUU AAA ACA at 5 nM A H16A(−06+25) UCU UUU CUA GAU Skipping CCG CUU UUA AAA at 5 nM CCU GUU A H16A(−06+19) CUA GAU CCG CUU UUA Skipping AAA CCU GUU A at 25 nM H16A(+87+109) CCG UCU UCU GGG UCA Skipping CUG ACU UA at 100 nM H16A(−07+19) CUA GAU CCG CUU UUA No  AAA CCU GUU AA skipping H16A(−07+13) CCG CUU UUA AAA CCU No  GUU AA skipping H16A(+12+37) UGG AUU GCU UUU UCU No  UUU CUA GAU CC skipping H16A(+92+116) CAU GCU UCC GUC UUC No  UGG GUC ACU G skipping H16A(+45+67) G AUC UUG UUU GAG No  UGA AUA CAG U skipping H16A(+105+126) GUU AUC CAG CCA No  UGC UUC CGU C skipping H16D(+05−20) UGA UAA UUG GUA UCA No  CUA ACC UGU G skipping H16D(+12−11) GUA UCA CUA ACC UGU No  GCU GUA C skipping

Antisense Oligonucleotides Directed at Exon 19

Antisense oligonucleotides directed at exon 19 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H19A(+35+65) [SEQ ID NO:79] induced substantial exon 19 skipping when delivered into cells at a concentration of 10 nM. This antisense molecule also showed very strong exon skipping at concentrations of 25, 50, 100, 300 and 600 nM.

FIG. 10 illustrates exon 19 and 20 skipping using a “cocktail” of antisense oligonucleotides, as tested using gel electrophoresis. It is interesting to note that it was not easy to induce exon 20 skipping using single antisense oligonucleotides H20A(+44+71) [SEQ ID NO:81] or H20A(+149+170) [SEQ ID NO:82], as illustrated in sections 2 and 3 of the gel shown in FIG. 10. Whereas, a “cocktail” of antisense oligonucleotides was more efficient as can be seen in section 4 of FIG. 10 using a “cocktail” of antisense oligonucleotides H20A(+44+71) and H20A(+149+170). When the cocktail was used to target exon 19, skipping was even stronger (see section 5, FIG. 10).

FIG. 11 illustrates gel electrophoresis results of exon 19/20 skipping using “weasels” The “weasels” were effective in skipping exons 19 and 20 at concentrations of 25, 50, 100, 300 and 600 nM. A further “weasel” sequence is shown in the last row of Table 3C. This compound should give good results.

Antisense Oligonucleotides Directed at Exon 20

Antisense oligonucleotides directed at exon 20 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

None of the antisense oligonucleotides tested induced exon 20 skipping when delivered into cells at a concentration of 10, 25, 50, 300 or 600 nM (see Table 15). Antisense molecules H20A(−11+17) [SEQ ID NO:86] and H20D(+08−20) [SEQ ID NO:87] are yet to be tested.

However, a combination or “cocktail” of H20A(+44+71) [SEQ ID NO: 81] and H20(+149+170) [SEQ ID NO:82] in a ratio of 1:1, exhibited very strong exon skipping at a concentration of 100 nM and 600 nM. Further, a combination of antisense molecules H19A(+35+65) [SEQ ID NO:79], H20A(+44+71) [SEQ ID NO:81] and H20A(+149+170) [SEQ ID NO:82] in a ratio of 2:1:1, induced very strong exon skipping at a concentration ranging from 10 nM to 600 nM.

TABLE 15 Antisense Ability  Oligo- to nucloetide induce name Sequence skipping H20A(+44+71) CUG GCA GAA UUC GAU No  CCA CCG GCU GUU C skipping H20A(+149+170) CAG CAG UAG UUG UCA No  UCU GCU C skipping H20A(+185+203) UGA UGG GGU GGU GGG No  UUG G skipping H20A(−08+17) AUC UGC AUU AAC ACC No  CUC UAG AAA G skipping H20A(+30+53) CCG GCU GUU CAG UUG No  UUC UGA GGC skipping H20A(−11+17) AUC UGC AUU AAC ACC Not  CUC UAG AAA GAA A tested yet H20D(+08−20) GAA GGA GAA GAG AUU Not  CUU ACC UUA CAA A tested yet H20A(+44+71) & CUG GCA GAA UUC GAU Very  H20A(+149+170) CCA CCG GCU GUU C strong CAG CAG UAG UUG UCA skipping UCU GCU C H19A(+44+71): GCC UGA GCU GAU Very  CUG CUG GCA UCU strong UGC AGU U skipping H20A(+44+71); CUG GCA GAA UUC GAU CCA CCG GCU GUU C H20A(+149+170) CAG CAG UAG UUG UCA UCU GCU C

Antisense Oligonucleotides Directed at Exon 21

Antisense oligonucleotides directed at exon 21 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H21A(+85+108) [SEQ ID NO:92] and H21A(+85+106) [SEQ ID NO:91] induced exon 21 skipping when delivered into cells at a concentration of 50 nM. Table 16 below includes other antisense molecules tested at a concentration range of 5, 25, 50, 100, 200 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping

TABLE 16 Antisense Ability to Oligonucleotide induce name Sequence skipping H21A(−06+16) GCC GGU UGA CUU Skips at CAU CCU GUG C 600 nM H21A(+85+106) CUG CAU CCA GGA Skips at ACA UGG GUC C 50 nM H21A(+85+108) GUC UGC AUC CAG   Skips at GAA CAU GGG UC 50 nM H21A(+08+31) GUU GAA GAU CUG   Skips AUA GCC GGU UGA faintly to H21D(+18−07) UAC UUA CUG UCU   No  GUA GCU CUU UCU skipping

Antisense Oligonucleotides Directed at Exon 22

Antisense oligonucleotides directed at exon 22 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 12 illustrates differing efficiencies of two antisense molecules directed at exon 22 acceptor splice site. H22A(+125+106) [SEQ ID NO:96] and H22A(+80+101) [SEQ ID NO: 98] induce strong exon 22 skipping from 50 nM to 600 nM concentration.

H22A(+125+146) [SEQ ID NO:96] and H22A(+80+101) [SEQ ID NO:98] induced exon 22 skipping when delivered into cells at a concentration of 50 nM. Table 17 below shows other antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed a variable ability to induce exon skipping.

TABLE 17 Antisense Ability to oligonucleotide induce name Sequence skipping H22A(+22+45) CAC UCA UGG UCU   No  CCU GAU AGC GCA skipping H22A(+125+146) CUG CAA UUC CCC   Skipping  GAG UCU CUG C to 50 nM H22A(+47+69) ACU GCU GGA CCC   Skipping  AUG UCC UGA UG to 300 nM H22A(+80+101) CUA AGU UGA GGU   Skipping  AUG GAG AGU to 50 nM H22D(+13−11) UAU UCA CAG ACC   No  UGC AAU UCC CC skipping

Antisense Oligonucleotides Directed at Exon 23

Antisense oligonucleotides directed at exon 23 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

Table 18 below shows antisense molecules tested at a concentration range of 25, 50, 100, 300 and 600 nM. These antisense molecules showed no ability to induce exon skipping or are yet to be tested.

TABLE 18 Antisense Ability to oligonucleotide induce name Sequence skipping H23A(+34+59) ACA GUG GUG CUG AGA   No  UAG UAU AGG CC skipping H23A(+18+39) UAG GCC ACU UUG UUG  No  CUC UUG C Skipping H23A(+72+90) UUC AGA GGG CGC UUU  No  CUU C Skipping

Antisense Oligonucleotides Directed at Exon 24

Antisense oligonucleotides directed at exon 24 were prepared using similar methods as described above. Table 19 below outlines the antisense oligonucleotides directed at exon 24 that are yet to be tested for their ability to induce exon 24 skipping.

TABLE 19 Antisense Ability to oligonucleotide induce name Sequence skipping H24A(+48+70) GGG CAG GCC AUU CCU  Needs  CCU UCA GA testing H24A(−02+22) UCU UCA GGG UUU GUA  Needs  UGU GAU UCU testing

Antisense Oligonucleotides Directed at Exon 25

Antisense oligonucleotides directed at exon 25 were prepared using similar methods as described above. Table 20 below shows the antisense oligonucleotides directed at exon 25 that are yet to be tested for their ability to induce exon 25 skipping.

TABLE 20 Antisense Ability to oligonucleotide induce name Sequence skipping H25A(+9+36) CUG GGC UGA AUU GUC Needs UGA AUA UCA CUG testing H25A(+131+156) CUG UUG GCA CAU GUG Needs AUC CCA CUG AG testing H25D(+16−08) GUC UAU ACC UGU UGG Needs CAC AUG UGA testing

Antisense Oligonucleotides Directed at Exon 26

Antisense oligonucleotides directed at exon 26 were prepared using similar methods as described above. Table 21 below outlines the antisense oligonucleotides directed at exon 26 that are yet to be tested for their ability to induce exon 26 skipping.

TABLE 21 Antisense Ability oligo- to nucleotide induce name Sequence skipping H26A(+132+156) UGC UUU CUG UAA  Needs UUC AUC UGG AGU testing U H26A(−07+19) CCU CCU UUC UGG    Needs CAU AGA CCU UCC testing AC H26A(+68+92) UGU GUC AUC CAU    Faint UCG UGC AUC UCU  skipping G at 600 nM

Antisense Oligonucleotides Directed at Exon 27

Antisense oligonucleotides directed at exon 27 were prepared using similar methods as described above. Table 22 below outlines the antisense oligonucleotides directed at exon 27 that are yet to be tested for their ability to induce exon 27 skipping.

TABLE 22 Antisense Ability to oligonucleotide induce name Sequence skipping H27A(+82+106) UUA AGG CCU CUU GUG   Needs  CUA CAG GUG G testing H27A(−4+19) GGG CCU CUU CUU UAG   Faint  CUC UCU GA skipping  at 600 and 300 nM H27D(+19−03) GAC UUC CAA AGU CUU   v. strong  GCA UUU C skipping at 600 and  300 nM

Antisense Oligonucleotides Directed at Exon 28

Antisense oligonucleotides directed at exon 28 were prepared using similar methods as described above. Table 23 below outlines the antisense oligonucleotides directed at exon 28 that are yet to be tested for their ability to induce exon 28 skipping.

TABLE 23 Antisense Ability to oligonucleotide induce name Sequence skipping H28A(−05+19) GCC AAC AUG CCC AAA   v. strong CUU CCU AAG skipping   at 600   and 300 nM H28A(+99+124) CAG AGA UUU CCU CAG   Needs  CUC CGC CAG GA testing H28D(+16−05) CUU ACA UCU AGC ACC   v. strong UCA GAG skipping  at 600 and 300 nM

Antisense Oligonucleotides Directed at Exon 29

Antisense oligonucleotides directed at exon 29 were prepared using similar methods as described above. Table 24 below outlines the antisense oligonucleotides directed at exon 29 that are yet to be tested for their ability to induce exon 29 skipping.

TABLE 24 Antisense Ability to oligonucleotide induce name Sequence skipping H29A(+57+81) UCC GCC AUC UGU   Needs UAG GGU CUG UGC  testing C H29A(+18+42) AUU UGG GUU AUC   v. strong CUC UGA AUG UCG  skipping at C 600 and  300 nM H29D(+17−05) CAU ACC UCU UCA   v. strong UGU AGU UCC C skipping at 600 and  300 nM

Antisense Oligonucleotides Directed at Exon 30

Antisense oligonucleotides directed at exon 30 were prepared using similar methods as described above. Table 25 below outlines the antisense oligonucleotides directed at exon 30 that are yet to be tested for their ability to induce exon 30 skipping.

TABLE 25 Antisense oligonucleotide name Sequence Ability to induce skipping H30A(+122+147) CAU UUG AGC UGC GUC CAC CUU Needs testing GUC UG H30A(+25+50) UCC UGG GCA GAC UGG AUG CUC Very strong skipping at UGU UC 600 and 300 nM. H30D(+19−04) UUG CCU GGG CUU CCU GAG GCA Very strong skipping at UU 600 and 300 nM.

Antisense Oligonucleotides Directed at Exon 31

Antisense oligonucleotides directed at exon 31 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 13 illustrates differing efficiencies of two antisense molecules directed at exon 31 acceptor splice site and a “cocktail” of exon 31 antisense oligonucleotides at varying concentrations. H31 D(+03−22) [SEQ ID NO:124] substantially induced exon 31 skipping when delivered into cells at a concentration of 20 nM. Table 26 below also includes other antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.

TABLE 26 Antisense oligonucleotide Ability to induce name Sequence skipping H31D(+06−18) UUC UGA AAU AAC AUA UAC CUG UGC Skipping to 300 nM H31D(+03−22) UAG UUU CUG AAA UAA CAU AUA CCU G Skipping to 20 nM H31A(+05+25) GAC UUG UCA AAU CAG AUU GGA No skipping H31D(+04−20) GUU UCU GAA AUA ACA UAU ACC UGU Skipping to 300 nM

Antisense Oligonucleotides Directed at Exon 32

Antisense oligonucleotides directed at exon 32 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H32D(+04−16) [SEQ ID NO:127] and H32A(+49+73) [SEQ ID NO:130] induced exon 32 skipping when delivered into cells at a concentration of 300 nM. Table 27 below also shows other antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules did not show an ability to induce exon skipping.

TABLE 27 Antisense oligonucleotide Ability to induce name Sequence skipping H32D(+04−16) CAC CAG AAA UAC AUA CCA CA Skipping to 300 nM H32A(+151+170) CAA UGA UUU AGC UGU GAC UG No skipping H32A(+10+32) CGA AAC UUC AUG GAG ACA UCU UG No skipping H32A(+49+73) CUU GUA GAC GCU GCU CAA AAU Skipping to 300 nM UGG C

Antisense Oligonucleotides Directed at Exon 33

Antisense oligonucleotides directed at exon 33 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 14 shows differing efficiencies of two antisense molecules directed at exon 33 acceptor splice site. H33A(+64+88) [SEQ ID NO:134] substantially induced exon 33 skipping when delivered into cells at a concentration of 10 nM. Table 28 below includes other antisense molecules tested at a concentration of 100, 200 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.

TABLE 28 Antisense oligonucleotide Ability to induce name Sequence skipping H33D(+09−11) CAU GCA CAC ACC UUU GCU CC No skipping H33A(+53+76) UCU GUA CAA UCU GAC GUC CAG UCU Skipping to 200 nM H33A(+30+56) GUC UUU AUC ACC AUU UCC ACU UCA Skipping to 200 nM GAC H33A(+64+88) CCG UCU GCU UUU UCU GUA CAA UCU G Skipping to 10 nM

Antisense Oligonucleotides Directed at Exon 34

Antisense oligonucleotides directed at exon 34 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

Table 29 below includes antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed a variable ability to induce exon skipping.

TABLE 29 Antisense oligonucleotide Ability to induce name Sequence skipping H34A(+83+104) UCC AUA UCU GUA GCU GCC AGC C No skipping H34A(+143+165) CCA GGC AAC UUC AGA AUC CAA AU No skipping H34A(−20+10) UUU CUG UUA CCU GAA AAG AAU UAU AAU Not tested GAA H34A(+46+70) CAU UCA UUU CCU UUC GCA UCU UAC G Skipping to 300 nM H34A(+95+120) UGA UCU CUU UGU CAA UUC CAU AUC UG Skipping to 300 nM H34D(+10−20) UUC AGU GAU AUA GGU UUU ACC UUU Not tested CCC CAG H34A(+72+96) CUG UAG CUG CCA GCC AUU CUG UCA AG No skipping

Antisense Oligonucleotides Directed at Exon 35

Antisense oligonucleotides directed at exon 35 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 15 shows differing efficiencies of antisense molecules directed at exon 35 acceptor splice site. H35A(+24+43) [SEQ ID NO:144] substantially induced exon 35 skipping when delivered into cells at a concentration of 20 nM. Table 30 below also includes other antisense molecules tested at a concentration of 100 and 300 nM. These antisense molecules showed no ability to induce exon skipping.

TABLE 30 Antisense oligonucleotide Ability to induce name Sequence skipping H35A(+141+161) UCU UCU GCU CGG GAG GUG ACA Skipping to 20 nM H35A(+116+135) CCA GUU ACU AUU CAG AAG AC No skipping H35A(+24+43) UCU UCA GGU GCA CCU UCU GU No skipping

Antisense Oligonucleotides Directed at Exon 36

Antisense oligonucleotides directed at exon 36 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

Antisense molecule H36A(+26+50) [SEQ ID NO:145] induced exon 36 skipping when delivered into cells at a concentration of 300 nM, as shown in FIG. 16.

Antisense Oligonucleotides Directed at Exon 37

Antisense oligonucleotides directed at exon 37 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 17 shows differing efficiencies of two antisense molecules directed at exon 37 acceptor splice site. H37A(+82+105) [SEQ ID NO:148] and H37A(+134+157) [SEQ ID NO:149] substantially induced exon 37 skipping when delivered into cells at a concentration of 10 nM. Table 31 below shows the antisense molecules tested.

TABLE 31 Antisense oligonucleotide Ability to induce name Sequence skipping H37A(+26+50) CGU GUA GAG UCC ACC UUU GGG CGU A No skipping H37A(+82+105) UAC UAA UUU CCU GCA GUG GUC ACC Skipping to 10 nM H37A(+134+157) UUC UGU GUG AAA UGG CUG CAA AUC Skipping to 10 nM

Antisense Oligonucleotides Directed at Exon 38

Antisense oligonucleotides directed at exon 38 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 18 illustrates antisense molecule H38A(+88+112) [SEQ ID NO:152], directed at exon 38 acceptor splice site. H38A(+88+112) substantially induced exon 38 skipping when delivered into cells at a concentration of 10 nM. Table 32 below shows the antisense molecules tested and their ability to induce exon skipping.

TABLE 32 Antisense oligonucleotide Ability to induce name Sequence skipping H38A(−01+19) CCU UCA AAG GAA UGG AGG CC No skipping H38A(+59+83) UGC UGA AUU UCA GCC UCC AGU GGU U Skipping to 10 nM H38A(+88+112) UGA AGU CUU CCU CUU UCA GAU UCA C Skipping to 10 nM

Antisense Oligonucleotides Directed at Exon 39

Antisense oligonucleotides directed at exon 39 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H39A(+62+85) [SEQ ID NO:153] induced exon 39 skipping when delivered into cells at a concentration of 100 nM. Table 33 below shows the antisense molecules tested and their ability to induce exon skipping.

TABLE 33 Antisense oligonucleotide Ability to induce name Sequence skipping H39A(+62+85) CUG GCU UUC UCU CAU CUG UGA UUC Skipping to 100 nM H39A(+39+58) GUU GUA AGU UGU CUC CUC UU No skipping H39A(+102+121) UUG UCU GUA ACA GCU GCU GU No skipping H39D(+10−10) GCU CUA AUA CCU UGA GAG CA Skipping to 300 nM

Antisense Oligonucleotides Directed at Exon 40

Antisense oligonucleotides directed at exon 40 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 19 illustrates antisense molecule H40A(−05+17) [SEQ ID NO:157] directed at exon 40 acceptor splice site. H40A(−05+17) and H40A(+129+153) [SEQ ID NO:158] both substantially induced exon 40 skipping when delivered into cells at a concentration of 5 nM.

Antisense Oligonucleotides Directed at Exon 42

Antisense oligonucleotides directed at exon 42 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 20 illustrates antisense molecule H42A(−04+23) [SEQ ID NO:159], directed at exon 42 acceptor splice site. H42A(−4+23) and H42D(+19−02) [SEQ ID NO:161] both induced exon 42 skipping when delivered into cells at a concentration of 5 nM. Table 34 below shows the antisense molecules tested and their ability to induce exon 42 skipping.

TABLE 34 Antisense oligonucleotide Ability to induce name Sequence skipping H42A(−4+23) AUC GUU UCU UCA CGG ACA GUG UGC Skipping to 5 nM UGG H42A(+86+109) GGG CUU GUG AGA CAU GAG UGA UUU Skipping to 100 nM H42D(+19−02) A CCU UCA GAG GAC UCC UCU UGC Skipping to 5 nM

Antisense Oligonucleotides Directed at Exon 43

Antisense oligonucleotides directed at exon 43 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H43A(+101+120) [SEQ ID NO:163] induced exon 43 skipping when delivered into cells at a concentration of 25 nM. Table 35 below includes the antisense molecules tested and their ability to induce exon 43 skipping.

TABLE 35 Antisense oligonucleotide Ability to induce name Sequence skipping H43D(+10−15) UAU GUG UUA CCU ACC CUU GUC GGU C Skipping to 100 nM H43A(+101+120) GGA GAG AGC UUC CUG UAG CU Skipping to 25 nM H43A(+78+100) UCA CCC UUU CCA CAG GCG UUG CA Skipping to 200 nM

Antisense Oligonucleotides Directed at Exon 44

Antisense oligonucleotides directed at exon 44 were prepared using similar methods as described above. Testing for the ability of these antisense molecules to induce exon 44 skipping is still in progress. The antisense molecules under review are shown as SEQ ID Nos: 165 to 167 in Table 1A.

Antisense Oligonucleotides Directed at Exon 45

Antisense oligonucleotides directed at exon 45 were prepared using similar methods as described above. Testing for the ability of these antisense molecules to induce exon 45 skipping is still in progress. The antisense molecules under review are shown as SEQ ID Nos: 207 to 211 in Table 1A.

Antisense Oligonucleotides Directed at Exon 46

Antisense oligonucleotides directed at exon 46 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 21 illustrates the efficiency of one antisense molecule directed at exon 46 acceptor splice site. Antisense oligonucleotide H46A(+86+115) [SEQ ID NO:203] showed very strong ability to induce exon 46 skipping. Table 36 below includes antisense molecules tested. These antisense molecules showed varying ability to induce exon 46 skipping.

TABLE 36 Antisense oligonucleotide Ability to induce name Sequence skipping H46D(+16−04) UUA CCU UGA CUU GCU CAA GC No skipping H46A(+90+109) UCC AGG UUC AAG UGG GAU AC No skipping H46A(+86+115) CUC UUU UCC AGG UUC AAG UGG GAU Good skipping ACU AGC to 100 nM H46A(+107+137) CAA GCU UUU CUU UUA GUU GCU GCU Good skipping CUU UUC C to 100 nM H46A(−10+20) UAU UCU UUU GUU CUU CUA GCC UGG Weak skipping AGA AAG H46A(+50+77) CUG CUU CCU CCA ACC AUA AAA CAA Weak skipping AUU C

Antisense Oligonucleotides Directed at Exon 47

Antisense oligonucleotides directed at exon 47 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

H47A(+76+100) [SEQ ID NO:170] and H47A(−09+12) [SEQ ID NO:172] both induced exon 47 skipping when delivered into cells at a concentration of 200 nM. H47D(+25−02) [SEQ ID NO: 171] is yet to be prepared and tested.

Antisense Oligonucleotides Directed at Exon 50

Antisense oligonucleotides directed at exon 50 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

Antisense oligonucleotide molecule H50(+02+30) [SEQ ID NO: 173] was a strong inducer of exon skipping. Further, H50A(+07+33) [SEQ ID NO:174] and H50D(+07−18) [SEQ ID NO:175] both induced exon 50 skipping when delivered into cells at a concentration of 100 nM.

Antisense Oligonucleotides Directed at Exon 51

Antisense oligonucleotides directed at exon 51 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 22 illustrates differing efficiencies of two antisense molecules directed at exon 51 acceptor splice site. Antisense oligonucleotide H51A(+66+90) [SEQ ID NO:180] showed the stronger ability to induce exon 51 skipping. Table 37 below includes antisense molecules tested at a concentration range of 25, 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon skipping. The strongest inducers of exon skipping were antisense oligonucleotide H51A(+61+90) [SEQ ID NO: 179] and H51A(+66+95) [SEQ ID NO: 179].

TABLE 37 Antisense Ability to oligonucleotide induce name Sequence skipping H51A(−01+25) ACC AGA GUA ACA GUC UGA GUA GGA GC Faint skipping H51D(+16−07) CUC AUA CCU UCU GCU UGA UGA UC Skipping at 300 nM H51A(+111+134) UUC UGU CCA AGC CCG GUU GAA AUC Needs re- testing H51A(+61+90) ACA UCA AGG AAG AUG GCA UUU CUA GUU Very strong UGG skipping H51A(+66+90) ACA UCA AGG AAG AUG GCA UUU CUA G skipping H51A(+66+95) CUC CAA CAU CAA GGA AGA UGG CAU UUC Very strong UAG skipping H51D(+08−17) AUC AUU UUU UCU CAU ACC UUC UGC U No skipping H51A/D(+08−17) AUC AUU UUU UCU CAU ACC UUC UGC UAG No skipping & (−15+?) GAG CUA AAA H51A(+175+195) CAC CCA CCA UCA CCC UCY GUG No skipping H51A(+199+220) AUC AUC UCG UUG AUA UCC UCA A No skipping

Antisense Oligonucleotides Directed at Exon 52

Antisense oligonucleotides directed at exon 52 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 22 also shows differing efficiencies of four antisense molecules directed at exon 52 acceptor splice site. The most effective antisense oligonucleotide for inducing exon 52 skipping was H52A(+17+37) [SEQ ID NO:188].

Table 38 below shows antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon 50 skipping. Antisense molecules H52A(+12+41) [SEQ ID NO:187] and H52A(+17+37) [SEQ ID NO:188] showed the strongest exon 50 skipping at a concentration of 50 nM.

TABLE 38 Antisense Ability to oligonucleotide induce name Sequence skipping H52A(−07+14) UCC UGC AUU GUU GCC UGU AAG No skipping H52A(+12+41) UCC AAC UGG GGA CGC CUC UGU UCC AAA Very strong UCC skipping H52A(+17+37) ACU GGG GAC GCC UCU GUU CCA Skipping to 50 nM H52A(+93+112) CCG UAA UGA UUG UUC UAG CC No skipping H52D(+05−15) UGU UAA AAA ACU UAC UUC GA No skipping

Antisense Oligonucleotides Directed at Exon 53

Antisense oligonucleotides directed at exon 53 were prepared and tested for their ability to induce exon skipping in human muscle cells using similar methods as described above.

FIG. 22 also shows antisense molecule H53A(+39+69) [SEQ ID NO:193] directed at exon 53 acceptor splice site. This antisense oligonucleotide was able to induce exon 53 skipping at 5, 100, 300 and 600 nM. A “cocktail” of three exon antisense oligonucleotides:—H53D(+23+47) [SEQ ID NO:195], H53A(+150+175) [SEQ ID NO:196] and H53A(+14−07) [SEQ ID NO:194], were also tested, as shown in FIG. 20 and exhibited an ability to induce exon skipping.

Table 39 below includes other antisense molecules tested at a concentration range of 50, 100, 300 and 600 nM. These antisense molecules showed varying ability to induce exon 53 skipping. Antisense molecule H53A(+39+69) [SEQ ID NO:193] induced the strongest exon 53 skipping.

TABLE 39 Antisense oligonucleotide Ability to induce name Sequence skipping H53A(+45+69) CAU UCA ACU GUU GCC UCC GGU UCU G Faint skipping at 50 nM H53A(+39+62) CUG UUG CCU CCG GUU CUG AAG GUG Faint skipping at 50 nM H53A(+39+69) CAU UCA ACU GUU GCC UCC GGU UCU Strong skipping GAA GGU G to 50 nM H53D(+14−07) UAC UAA CCU UGG UUU CUG UGA Very faint skipping to 50 nM H53A(+23+47) CUG AAG GUG UUC UUG UAC UUC AUC C Very faint skipping to 50 nM H53A(+150+176) UGU AUA GGG ACC CUC CUU CCA UGA Very faint CUC skipping to 50 nM H53D(+20−05) CUA ACC UUG GUU UCU GUG AUU UUC U Not made yet H53D(+09−18) GGU AUC UUU GAU ACU AAC CUU GGU Faint at 600 nM UUC H53A(−12+10) AUU CUU UCA ACU AGA AUA AAA G No skipping H53A(−07+18) GAU UCU GAA UUC UUU CAA CUA GAA U No skipping H53A(+07+26) AUC CCA CUG AUU CUG AAU UC No skipping H53A(+124+145) UUG GCU CUG GCC UGU CCU AAG A No skipping 

1-14. (canceled)
 15. An isolated antisense oligonucleotide of 25 to 50 nucleotides in length comprising SEQ ID NO: 180, wherein the uracil bases are optionally thymine bases.
 16. The antisense oligonucleotide of claim 15, wherein the antisense oligonucleotide comprises a non-natural backbone.
 17. The antisense oligonucleotide of claim 15, wherein the antisense oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.
 18. The antisense oligonucleotide of claim 15, wherein the antisense oligonucleotide does not activate RNase H.
 19. The antisense oligonucleotide of claim 16, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties.
 20. The antisense oligonucleotide of claim 19, wherein the non-natural moieties are morpholinos.
 21. The antisense oligonucleotide of claim 20, wherein the uracil bases are thymine bases.
 22. The antisense oligonucleotide of claim 15, wherein the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.
 23. The antisense oligonucleotide of claim 22, wherein the non-natural inter-nucleotide linkages are modified phosphates.
 24. The antisense oligonucleotide of claim 23, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.
 25. The antisense oligonucleotide of claim 24, wherein the modified phosphates are phosphoroamidates.
 26. The antisense oligonucleotide of claim 24, wherein the modified phosphates are phosphoromorpholidates.
 27. The antisense oligonucleotide of claim 15, wherein the sugar moieties of the oligonucleotide backbone are replaced with non-natural moieties and the inter-nucleotide linkages of the oligonucleotide backbone are replaced with non-natural inter-nucleotide linkages.
 28. The antisense oligonucleotide of claim 27, wherein the non-natural moieties are morpholinos and the non-natural internucleotide linkages are modified phosphates.
 29. The antisense oligonucleotide of claim 28, wherein the modified phosphates are methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates or phosphoroamidates.
 30. The antisense oligonucleotide of claim 29, wherein the modified phosphates are phosphoroamidates.
 31. The antisense oligonucleotide of claim 29, wherein the modified phosphates are phosphoromorpholidates.
 32. The antisense oligonucleotide of claim 29, wherein the uracil bases are thymine bases.
 33. The antisense oligonucleotide of claim 32, wherein the antisense oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.
 34. The antisense oligonucleotide of claim 15, wherein the antisense oligonucleotide is 25 nucleotides in length.
 35. The antisense oligonucleotide of claim 34, wherein the uracil bases are thymine bases.
 36. A pharmaceutical composition, comprising the antisense oligonucleotide of claim 15 and a pharmaceutically acceptable carrier.
 37. The pharmaceutical composition of claim 36, wherein the pharmaceutically acceptable carrier is phosphate-buffered saline.
 38. A pharmaceutically acceptable salt of the antisense oligonucleotide of claim
 15. 